Assay of isomerised and/or optically inverted proteins and protein fragments

ABSTRACT

In an in vitro diagnostic test for osteoarthritis or rheumatoid arthritis, the amount or presence in a sample of anisomerised or optically inverted protein fragment is measured which derives from perlecan, bigylcan, decorin, fibrillin-1 or protocadherin or which is a specific sequence from aggrecan, type II collagen, COMP or CILP.

[0001] The present invention relates to immunoassays for non-collagen cartilage proteins and their fragments in biological samples such as body fluids. Such proteins and protein fragments may serve as an index of joint disease.

[0002] A number of diseases are associated with increased turnover of the extra cellular matrix of tissues or organs in the mammalian body. As examples of such diseases or pathological conditions, arthritic diseases involves increased turnover of tissues of the joint, osteoporosis is associated with increased turnover of bone tissue, psoriasis and scleroderma is associated with increased turnover of skin and various cancer types are also associated with increased tissue turnover of the disease affected tissue(s) or organ(s). Of special relevance in these conditions is a quantification of the catabolic processes associated with the disease as these often predominates and results in degradation of the affected tissues or organs. Such catabolic process can be difficult to quantify, and specific biochemical markers for monitoring such processes would be of great clinical utility for diagnosis, monitoring and assessing prognosis of the diseases. Furthermore, such markers could be used in pre-clinical and clinical research for identification and assessment of new therapeutic agents and for optimising treatment dose and treatment modality. However, even though a need for such markers clearly exists it has been difficult to identify and develop such biochemical markers and very few such markers exist today for routine use in clinical management of disease or research into disease mechanisms and development of new therapeutic agents.

[0003] The present invention provides a general method for identification of biochemical markers specific for catabolic processes in a mammalian tissue or organ. This method has been applied for identification of new markers of cartilage turnover; a process which is of relevance for all arthritic diseases, as described below.

[0004] Increased awareness of the early biochemical and structural changes in cartilage-related diseases in combination with the introduction of new disease suppressive agents has created the need to develop improved tools to assess disease severity and prognosis. Thus the need for sensitive simple and reliable markers for cartilage degradation is evident, and such markers will fulfil important clinical purpose for management of arthritic disease.

[0005] As of today, no tissue specific biochemical marker exists which is of general use for diagnosis and monitoring of cartilage degradation in Rheumatoid Arthritis (RA) and Osteoarthritis (OA) patients. Markers such as C-reactive protein (CRP), rheumatoid factor (RF) and erythrocyte sedimentation rate (ESR) are frequently applied in RA. These markers provide information about the inflammatory reactions in the disease, but are not specific for joint diseases, and in spite of their wide-spread use, their clinical utility is still discussed. At present one of the best ways to obtain information about the status of the (individual) joints in arthritis patients is radiological examination.

[0006] Measurement of metabolites, such as hyaluronates and aggrecan fragments arising from destruction of the specific organ (the joints) affected by the arthritis has been reported, for review please see Wollheim (Woliheim, 1996). The two main arthritic diseases, RA and OA are briefly described below.

[0007] Osteoarthritis:

[0008] Osteoarthritis (OA) is a chronic disease characterized by degenerative changes of the joints and bone with destruction of the cartilage and reactive formation of bone in the periphery of the joint. OA is a common disease, with an increasing incidence with age. At the age of 50 approximately 20% have symptoms of OA and radiologically almost 90% exhibit OA-related changes.

[0009] One of the earliest events that can be detected histologically is the loss of proteoglycans (aggrecan) from the tissue (Flannerly et al 1992). The likely mechanism is the fragmentation of aggrecan by proteinases. The generation of aggrecan fragments is detrimental to the proper functioning of cartilage. In later stages of the disease also collagen fibrils are degraded (Eyre et al 1991) and the surface of the cartilage is eroded due to loss of tissue (Inerot & Heineg{dot over (a)}rd, 1982).

[0010] Rheumatoid Arthritis:

[0011] Rheumatoid arthritis (RA) is an autoimmune disease, where the patients own immune system attacks the joints, causing inflammation and subsequent degradation of the cartilage matrix. The disease affects approximately 1% of the population. At present the underlying causes responsible for initiation of the disease are unknown, however studies of its epidemiology show that genetic as well as environmental factors contribute to the pathogenesis.

[0012] RA is characterized by chronic inflammation associated with erosion of both cartilage and bone. In advanced late stages of the disease, radiographs show characteristic evidence of cartilage loss and bone erosion at the joint margin. However much damage may be done early in the disease, before radiographs change. Sensitive indices of the current cartilage and bone changes in RA would therefore be of potential value to clinicians.

[0013] The present invention describes methods for identifying such markers and also methods for developing and applying assays for such markers for management of arthritic disease as well as for developing new therapeutic or disease management methods for arthritic disease.

[0014] One aim of the present invention is to provide new methods and procedures for identifying potential markers of cartilage degradation based on identification of isomerised and/or optically inverted fragments of cartilage-derived proteins. Such markers will provide a significant advantage for the clinical management of OA, RA as well as other diseases affecting joint metabolism. Furthermore the use of such markers for management of arthritic disease and development of anti-arthritic therapeutic agents is described.

[0015] The Extracellular Matrix of Cartilage:

[0016] Cartilage matrix is synthesized, degraded, organized and maintained by a sparse population of chondrocytes (making up 0.5-2% of the total cartilage volume) (Poole et al 1994, Poole & Dieppe 1994). These cells are protected from the potentially damaging forces of mechanical function by the extra-cellular matrix (ECM) they produce.

[0017] The properties of cartilage are critically dependent upon the structure and integrity of the ECM. Thus, normal turnover is a conservative process in which the rate of matrix degradation does not exceed the rate at which it is replaced. There is a slow constant turnover of the ECM with chondrocytes both degrading and synthesizing matrix proteins. Healing of cartilage is dependent entirely on surviving chondrocytes near the margins of the injury. In adults these cells mediate essentially no repair.

[0018] A number of cartilage proteins have been identified and characterized. The main structural component of cartilage is made up of collagen fibers, predominantly composed of collagen type II with minor amounts (typically less than 10%) of collagen type IX and type XI. Interspaced between the collagen network are long chans of the negatively charged polysaccharide hyaluronic acids, to which several large proteoglycans are attached. The major proteoglycan is aggrecan. Aggrecan is a large protein with a molecular mass of 2600 kDa with a core protein of 220 kDa (Doege et al 1991). The protein is composed of 93% glycosaminoglycans (about 87% chondroitin sulphate and 6% keratan sulphate) and 7% protein. Aggrecan is a major component of cartilage where it accounts for about 10% of the dry weight of cartilage. The oligosaccharide moieties of aggrecan are highly negatively charged which draws water into the tissue as it creates a large osmotic swelling pressure. The water thus swells and expands the aggrecan-rich matrix. This places the collagen network under tension and an equilibrium is achieved when tension in the collagen network balances the swelling pressure (i.e., when no more water enters the tissue because the force is insufficient to stretch the collagen network any further). The aggrecan molecule is made up of 7 different domains. At the N-terminus there are two structurally related globular domains termed G1 and G2, separated by a short region known as the interglobular domain (IGD). Although the function of the G2 domain has not been determined the G1 domain is known to serve as a ‘linker’ to hyaluronan and is responsible for the formation of large aggrecan aggregates (Hardingham & Muir, 1972). The G1 domain is also in contact with cartilage link protein (see below). C-terminally to the G2 domain there is a long region consisting of two glycosaminoglycan-rich regions. The first is a domain rich in keratan sulphate (KS), whereas the other is composed of two domains rich in chondroitin sulphate (CS). At the C-terminus aggrecan possesses a third globular domain G3. The G3 domain seems to be lost soon after synthesis and secretion, the consequence of loss of the G3 domain is not yet understood (Hardingham & Fosang, 1992). Current evidence indicates that aggrecan fragments released during degradation of the cartilage matrix are heterogeneous products and may reflect various stages of disease progression depending on which fragments are monitored. In early or non-cartilage destructive diseases no or only small amounts of fragments related to the G1 domain are released (Saxne & Heineg{dot over (a)}rd 1992). Increased release of G1 related fragments, only accompanies the more profound and presumably irreversible cartilage derangement seen in patients with more damaged joints (Saxne & Heineg{dot over (a)}rd 1995).

[0019] A number of non-collagen cartilage proteins have been described although not all of these proteins are specific in cartilage, they play important structural and/or functional roles in the tissue, and may potentially serve as markers of cartilage turnover.

[0020] Cartilage intermediate layer protein (CILP) is noncollagenous cartilage protein composed of a single polypeptide chain with a molecular weight of 91.5 kDa, including N-linked oligosaccharides (Lorenzo et al. 1998a and 1998b). The protein is synthesized by chondrocytes and located to the interterritorial cartilage. It is neither found in the superficial nor deepest regions of the articular cartilage. CILP has been reported to increase with age and has been suggested to be a marker of early OA.

[0021] Cartilage Oligomeric Matrix Protein (COMP) is a non-collagenous extra-cellular matrix protein found predominantly in cartilage, but also in tendon, ligament and meniscus (Muller et al 1998). Several mesenchymal cells including synoviocytes and dermal fibroblasts produce substantial amounts of COMP (Dodge et al 1998). The physiological function of the protein is not known. COMP is a large disulfide-linked pentameric protein with a molecular weight of each monomer of about 100 kDa. The protein belongs to the thrombospondin family and it displays a high homology to thrombospondin 1-4 (Adams & Tucker 2000). In vitro data indicate that COMP may mediate cell binding within the cartilage matrix in accordance with the function of other members of the thrombospondin family. Furthermore, it is likely that COMP participates in regulation of collagen fibril formation (Rosenberg et al 1998). The major source of COMP within the joint appears to be fibroblastic cells in sub-synovial tissue. In cartilage COMP may be found as both the intact pentamer and in various fragments (43-160 kDa) generated by the action of MMP's as well as other proteases (Saxne & Heineg{dot over (a)}rd 1992, Ganu et al 1998). In OA cartilage the proportion of degraded COMP is higher than in normal cartilage even though the total amounts of COMP appears to be similar (Vilim et al 1997, Niedhart et al 1997). In RA cartilage there is a net loss of COMP, and the COMP fragments appear to be of smaller size than those seen in OA (Clarke et al 1999, Vingsbo-Lundberg et al 1998).

[0022] A number of COMP assays based on both polyclonal and monoclonal antibodies have been described in the literature, but the exact epitope specificity of the assays is generally not characterized (Saxne & Heineg{dot over (a)}rd 1992, Niedhart et al 1997). A number of cross-sectional studies have demonstrated elevated COMP levels in patients with OA, RA and other diseases affecting joint metabolism (Sharif et al 1995, Vingsbo-Lundberg et al 1998, Clarke et al 1999, Niedhart et al 1997).

[0023] Perlecan is a heparin sulphate proteoglycan that is expressed in all basement membranes, cartilage and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Relatively high levels of perlecan have been found in mature cartilage, and in vitro experiments have suggested that perlecan supports chondrocyte differentiation (Costell et al 1999).

[0024] Biglycan and decorin belong to the family of small leucine rich proteoglycans. They are relatively highly expressed in foetal cartilage, but are found in lower amounts in adult cartilage. Their functional role in cartilage is not fully elucidated but they may be involved in maintaining the structural integrity of the collagen fiber network and the hyaluronic acid mesh (Knudson & Knudson 2001). Furthermore they may play a role in chondrogenesis and cartilage degradation.

[0025] Fibrillin-1 (Fib-1) is a connective tissue protein, with an estimated molecular mass of 350,000 D. Fibrillin 1 is found in extracellular microfibrils in a variety of connective tissues. The protein has a widespread distribution in the connective tissue matrices of skin, lung, kidney, vasculature, cartilage, tendon, muscle, cornea, and ciliary zonule (Sakai et al 1986). The functional relationships between this glycoprotein and other components of the microfibrils and elastic fibers are unknown. Synthesis of fibrillin-1 correlates with late morphogenesis and the appearance of well-defined organ structures (Zhang and Ramirez 1995). It is widely expressed in developing limbs and digits, especially in the perichondrium (Keene et al 1997). Fib-1 appears as a loose meshwork of fibers within cartilage matrix by 20 weeks of fetal gestation. Until early adolescence, Fib-1 forms loose bundles of microfibrils within cartilage (Keene et al; 1997). However, by late adolescence, broad banded fibers composed of Fib-1 are found accumulated pericellularly within cartilage probably as laterally packed and crosslinked microfibrils. It has been proposed that fibrillin-1 provides force-bearing structural support to extracellular microfibrils and that it may growth-regulating functions in the perichondrium (Keene et al. 1997, Zhang and Ramirez 1995).

[0026] Protocadherins constitute a large family belonging to the cadherin superfamily and function in different tissues of a wide variety of multicellular organisms. Protocadherins have unique features that are not found in classic cadherins. Little is known about the Protocadherin subfamily gamma.

[0027] In joint diseases, the rate of degradation of matrix often exceeds the rate of synthesis, and, as consequence, the tissue becomes thin and mechanically weak (Heineg{dot over (a)}rd et al 1999). As the catabolic processes dominate in the arthritic disease states, fragments of cartilage proteins is produced, and such fragments can be measured in synovial fluid, serum and urine as markers of the cartilage degradation.

[0028] Joint diseases and related conditions involving abnormalities in the metabolism of cartilage cause widespread disability. Erosion of the articular cartilage is a typical finding in degenerative and inflammatory joint diseases, such as OA and RA.

[0029] Hypothesis Underlying the Invention

[0030] This invention describes a novel approach for identifying markers of cartilage degradation, and for development of diagnostic and prognostic assays for monitoring joint diseases. This approach is applicable not only for the identification of cartilage derived biochemical markers, but also as a generally applicable method for identification of proteins or protein fragments of an extra-cellular matrix tissue or organ of a mammal that may be suited as markers for turnover of the specific tissue or organ. We show that specific components of articular cartilage are prone to isomerisation and optical inversion (Example 1) and we identify specific isomerisation/optical inversion prone sites in several cartilage proteins. We also demonstrate that isomerised and/or optically inverted fragments of cartilage protein are found in circulation, and that measurements of such fragments provide an index of joint cartilage degradation.

[0031] Aspartic acid and asparagine (Asx) and glutamic acid and glutamine (Glx) residues will in some susceptible proteins undergo a spontaneous re-arrangement where the normal peptide bond between the Asx or Glx residue and the adjacent residue is transferred from the normal α-carboxyl group to the β-carboxyl group (α-carboxyl group for the Glx residues) of the side chain (Clarke 1987). The isomerisation reaction proceeds via a succinimide intermediate, which upon spontaneous hydrolysis may result in one of four forms: the normally occurring αL, the isoform βL, or the two optically inverted forms αD and βD as outlined in the following reaction scheme for aspartic acid—glycine (The reaction occurs analogously for other susceptible Asx and Glx containing sequences):

[0032] The above reaction scheme shows the authentic αL (A) form of the peptide bond as well as the three isomerised and/or optically inverted forms (βL (C), αD (G) and βD (I). The attack by the peptide backbone nitrogen on the side chain carbonyl group of an adjacent aspartyl residue can result in the formation of a succinimide ring, (A→B). The succinimide ring is prone to hydrolysis and optical inversion yielding peptides and iso-peptides in both the D and L configurations. Optical inversion proceeds through a carbanion intermediate (D, E and F) either through direct proton abstraction (A⇄D⇄G or C⇄F⇄I) or via the succinimide pathway (B⇄E⇄H). Throughout the figure the peptide backbone is shown as a bold line.

[0033] Cyclic imide formation (and thus isomerisation/optical inversion) can only occur, where the three-dimensional structure surrounding the Asx (asparagine or aspartic acid) or Glx (Glutamine or glutamic acid) has an optimal conformation and sufficient flexibility (Clarke 1987). Isomerisation and optical inversion via the succinimide intermediate as outlined above is a spontaneous reaction occurring with a slow rate under physiological conditions (Geiger & Clarke 1987, Fledelius et al 1997). As for all chemical reactions, increasing temperature can accelerate the reaction speed.

[0034] The introduction of such structural changes in a protein or peptide has profound effects on its function, stability and physical and chemical properties. Among other properties, the proteolytic degradation of proteins and peptides containing isomerised and/or optically inverted peptide linkages is significantly reduced compared to proteins and peptides composed exclusively of αL amino acids (Rafferty et al. 1988). Thus, protein fragments containing such modifications are not degraded to the same extent during normal tissue turnover (Van Regenmortel & Muller 1998) and they are much more likely to be present in circulation in measurable concentrations. Furthermore, by measuring proteins, or protein fragments containing isomerised and/or optically inverted peptide linkages, newly synthesized molecules will not contribute to the measurements, and it will thus reflect ongoing degradation processes.

[0035] In WO01/38872 (not published at the priority date hereof) we demonstrated that articular cartilage, a tissue with a very slow metabolism, contain proteins which are subject to isomerisation and optical inversion and we demonstrated that measurement of these proteins, or fragments thereof, can provide an index of joint cartilage degradation of diagnostic potential for assessing and monitoring joint diseases such as RA and OA.

[0036] We furthermore provided a method which is of general use for identification of markers of catabolic processes in mammalian tissues or organs, based on identification of isomerisation and/or optical inversion sites in extra-cellular matrix proteins and development of diagnostic assays specific for such isomerised and/or optically inverted proteins or protein fragments. By measuring protein fragments containing isomerised or optically inverted residues contributions from anabolic or tissue formation processes is minimized. We have found that isomerised and/or optically inverted fragments of aggrecan as well as collagen type I are found in serum, and that the measurements of such fragments reflect cartilage and bone resorption respectively.

[0037] Thus the invention described in WO01/38872 provided a novel approach to identification of biochemical markers of catabolic processes in mammalian tissues or organs, different from previous markers which have been reported as potential markers of i.e. arthritic disease such as YKL-40 (Johansen et al 1993, Johansen et al 1996, Harvey et al 1998), aggrecan (Heineg{dot over (a)}rd et al 1985) or cartilage oligomeric matrix protein (COMP) (Niedhart et al 1997, Saxne et al 1992, Saxne et al 1996). These markers rely on measurement of cartilage proteins in synovial fluid or serum, but as these cartilage proteins can be released during both anabolic and catabolic processes in the cartilage tissue it is not known whether they provide a specific measure of cartilage degradation. By measuring isomerised and/or optically inverted fragments of extra-cellular matrix derived proteins it is ensured that the marker is derived from tissue degradation as the isomerisation process occurs with relatively slow kinetics, and thus only in aged proteins significant isomerisation/optical inversion will have occurred (Radkiewicz et al 1996). Thus, no contribution from tissue formation (anabolic) processes is influencing measures of markers developed from isomerised and/or optically inverted extra-cellular matrix proteins.

[0038] The proteins covered by this previous application were aggrecan, cartilage link protein (CLP), cartilage oligomeric protein (COMP) and cartilage intermediate layer protein (CILP).

[0039] The present invention now provides in a first aspect a method of assay, comprising measuring in a biological sample the amount or presence of an isomerised and/or optically inverted protein or of one or more isomerised and/or optically inverted fragments from such a protein, wherein said protein is perlecan (SEQ ID NO 1), biglycan (SEQ ID NO 5), decorin (SEQ ID NO 3), fibrillin-1 (SEQ ID NO 10), or protocadherin (SEQ ID NO 8).

[0040] The SEQ ID NO's given above are only meant as examples of these proteins, homologous sequences from other mammals also lie within the scope of the present invention.

[0041] In a preferred embodiment, the method is based on the competitive binding of a isomerised and/or optically inverted protein or protein fragments produced in vivo or in vitro upon tissue degradation in biological fluids (body fluids or cell culture fluids) and of synthetic peptides essentially derived from such molecules to immunological binding partners.

[0042] In second preferred embodiment, the method is carried out using an immunological binding partner specific for a fragment containing an isomerised or optically inverted residue.

[0043] For purposes of the present invention, as disclosed and claimed herein, the following terms are defined below:

[0044] Antibody: a monoclonal or polyclonal antibody or immunoreactive fragment thereof (i.e. capable of binding the same antigenic determinant), including—but not limited to—Fab, Fab′ and F(ab′)₂ fragments.

[0045] *Asx: denotes one of the isomerised or optically inverted forms of aspargine or aspartic acid, which are αD-Asp, αD-Asn, βL-Asp or βD-Asp.

[0046] *Glx: denotes one of the isomerised or optically inverted forms of glutamine or glutamic acid, which are αD-Glu, αD-Gln, γL-Glu or γD-Glu.

[0047] Isomerised residues: Amino acid residues within a (poly)peptide where said residues are bound to their adjacent residue through an isopeptide bond (e.g. a bond proceeding through the side-chain β or γ-carboxyl group).

[0048] Optically inverted residues: D-amino acid residues.

[0049] Isomerised (about antigens, peptides, proteins and sequences): Molecules containing isomerised residues (isopeptide bonds).

[0050] Optically inverted (about antigens, peptides, proteins and sequences): Molecules containing D-amino acid residues.

[0051] Native (about antigens, peptides, proteins and sequences): Antigens/peptides/proteins and sequences composed of L-amino acid residues linked together by normal peptide bonds.

[0052] Test kit: A combination of reagents and instructions for use in conducting an assay.

[0053] Essentially derived (about structures): Structures with similar antigenicity, i.e. with an ability, above the level of a non-related peptide, to inhibit the binding of any of the mentioned synthetic peptides to an immunological binding partner immunoreactive with said synthetic peptide.

[0054] Biological fluid: Body fluids including urine, blood, serum, plasma saliva, sweat and synovial fluid, as well as fluids derived from cells in culture (e.g. supernatants from bone and cartilage cell cultures).

[0055] It is contemplated that the method may be used for assaying isopeptide or optically inverted fragments in biological fluids. It can also be used during pre-clinical and clinical testing of drugs to assess the impact of these drugs on metabolism and arthritic disease.

[0056] A method according to the invention may preferably determine the amount or presence of at least one of the above mentioned *Asx or *Glx containing proteins or protein fragments in said biological sample, wherein *Asx is αD-Asp, αD-Asn, βL-Asp or βD-Asp and *Glx is αD-Glu, αD-Gln, γL-Glu or γD-Glu.

[0057] Such a method may measure the amount of at least one protein or protein fragment containing the perlecan derived amino acid sequence YPVRIESSSASLANGHTL (SEQ ID NO 2) or a fragment thereof containing the E and/or N residue, wherein N denote αL-Asn or an amino acid covered by the term *Asx and E denote αL-Glu or an amino acid covered by the term *Glx (although in view of the requirement that the detected material is isomerised or opticlally inverted, N will not denote αL-Asn when E denotes αL-Glu).

[0058] Such a method may measure the amount of a least one protein or protein fragment containing the decorin derived amino acid sequence IADTNITSIPQGLPPSLTELLDG (SEQ ID NO 4) or a fragment thereof containing the D, E, N and/or Q residue, wherein N denotes αL-Asn or an amino acid covered by the term *Asx, D denotes αL-Asp or an amino acid covered by the term *ASX, E denotes αL-Glu or an amino acid covered by the term *Glx and Q denotes αL-Gln or an amino acid covered by the term *Glx (but for similar reasons not all will be αL).

[0059] Such a method may measure the amount of at least one protein or protein fragment containing the biglycan derived amino acid sequence FTLDDGPFMMNDE (SEQ ID NO 6) or a fragment thereof containing the D and/or E residue, wherein D denotes L-Asp or an amino acid covered by the term *Asx and E denotes L-Glu or an amino acid covered by the term *Glx (again not all four will be αL).

[0060] Such a method may measure the amount of at least one protein or protein fragment containing the protocadherin derived amino acid sequence YEQFRDLELRVIARDS (SEQ ID NO 8) or a fragment thereof containing the D and/or E residue, wherein D denotes αL-Asp or an amino acid covered by the term *AsX and E denotes αL-Glu or an amino acid covered by the term *Glx (all four will not be αL in the same detected molecule).

[0061] Such a method may measure the amount of at least one protein or protein fragment containing the fibrillin-1 derived amino acid sequence YEQFSGGCQDINE (SEQ ID NO 11) or a fragment thereof containing the D, E, N and/or Q residue, wherein N denotes αL-Asn or an amino acid covered by the term *Asx, D denotes αL-Asp or an amino acid covered by the term *Asx, E denotes αL-Glu or an amino acid covered by the term *Glx and Q denotes αL-Gln or an amino acid covered by the term *Glx (again not all will be in the αL form).

[0062] Such a method may measure the amount of at least one protein or protein fragment containing the protocadherin derived amino acid sequence YEQFRDLELR (SEQ ID NO 9) or a fragment thereof containing the D and/or E residue, wherein D denotes αL-Asp or an amino acid covered by the term *Asx and E denotes αL-Glu or an amino acid covered by the term *Glx (but not all will be αL).

[0063] In an alternative aspect, the invention includes a method of assay, comprising measuring in a sample the amount or presence of an isomerised or optically inverted fragment of a protein, wherein

[0064] (a) said protein is aggrecan and said fragment contains one of the amino acid sequences: LYPNQTGLPDPLSR (SEQ ID NO 12), SAIIATEQLQAAYEDGFHQC (SEQ ID NO 13), LATTGQLYLAWQAGMDM (SEQ ID NO 14), TGEDFVDIPENFFGV (SEQ ID NO 15), TGEDFVDIPEN (SEQ ID NO 16) or VSLPNYPAIPSDATLEVQSLRSNDSGVYR (SEQ ID NO 17) or a fragment thereof containing the D, E, N and/or Q residue, wherein N denotes αL-Asn or an amino acid covered by the term *Asx, D denotes αL-Asp or an amino acid covered by the term *Asx, E denotes αL-Glu or an amino acid covered by the term *Glx and Q denotes αL-Gln or an amino acid covered by the term *Glx.;

[0065] (b) said protein is type II collagen and said fragment contains the amino acid sequence EGSXGADGPXGRDG (SEQ ID NO 18) or a fragment thereof containing the D and/or E residue, wherein D denotes αL-Asp or an amino acid covered by the term *Asx and E denotes αL-Glu or an amino acid covered by the term *Glx;

[0066] (c) said protein is COMP and said fragment contains the amino acid sequence AQEDSDH (SEQ ID NO 19) or a fragment thereof containing the D, E and/or Q residue, wherein D denotes αL-Asp or an amino acid covered by the term *Asx, E denotes αL-Glu or an amino acid covered by the term *Glx and Q denotes αL-Gln or an amino acid covered by the term *Glx.; or

[0067] (d) said protein is CILP and said fragment contains the amino acid sequence LLTQTDSDGR (SEQ ID NO 20) or a fragment thereof containing the D and/or Q residue, wherein D denotes αL-Asp or an amino acid covered by the term *Asx and Q denotes αL-Gln or an amino acid covered by the term *Glx.

[0068] Fragments for detection as described above may preferably contain at least 4 amino acids, more preferably at least 8 amino acids.

[0069] In all of the above sequences, the amino acids marked by bold type face are of particular interest as sites of isomerisation or optical inversion. Only one of these sites need to be in an isomerised or optically inverted state in order to carry out the present invention.

[0070] The measurement performed in the methods of the present invention maybe carried out using an immunological binding partner which specifically binds an amino acid sequence comprising *Asx or *Glx.

[0071] Said immunological binding partner may be an antibody raised against a synthetic peptide having an amino acid sequence comprising *Asx or *Glx, or fragment of such an antibody having immunological binding specificity.

[0072] Preferably, said amino acid sequence corresponds to a characteristic sequence of a protein or fragment thereof covered by the present invention, with *Asx or *Glx substituting αL-Asp, -Asn, -Gln, or -Glu in said protein sequence.

[0073] Said measurement may provide an index of cartilage turnover relevant for conditions and diseases affecting joint tissue turnover.

[0074] Optionally the method further comprises carrying out a measurement of a second index of joint disease and determining the value of a parameter mathematically combining said two indices.

[0075] Thus the invention includes the use of ratios between measurements for monitoring a pathological situation or therapeutic intervention in a mammal, applying two or more assays specific for isomerised and/or optically inverted proteins or protein fragments from an extracellular matrix protein as described in the above.

[0076] The ratios can be generated from any such marker consisting of an isomerised and/or optically inverted protein or protein fragment from an extracellular matrix protein measured by the assays as described and other markers of tissue metabolism and/or tissue function for monitoring a pathological situation or therapeutic intervention in a mammal.

[0077] The invention includes the use of an isomerised and/or optically inverted protein or of one or more isomerised and/or optically inverted fragments from such a protein in an in vitro method of assay for the diagnosis or the assessment of the severity of OA or RA, comprising measuring in a biological sample the amount or presence of an isomerised and/or optically inverted protein or of one or more isomerised and/or optically inverted fragments from such a protein, wherein said protein is perlecan, biglycan, decorin, fibrillin-1, or protocadherin.

[0078] The invention include the use of an immunological binding partner which specifically binds an amino acid sequence comprising *Asx or *Glx flanked by amino acid residues of perlecan, biglycan, decorin, fibrillin-1, or protocadherin in an in vitro method for use in the diagnosis or the assessment of the severity of OA or RA.

[0079] In another aspect, the invention provides an immunological binding partner which specifically binds an amino acid sequence comprising *Asx or *Glx flanked by amino acid residues of perlecan, biglycan, decorin, fibrillin-1 or protocadherin.

[0080] The immunological binding partner may specifically bind a sequence set out above.

[0081] The invention includes a cell line producing a monoclonal antibody which is an immunological binding partner described herein.

[0082] The invention further includes a peptide of up to 20 (e.g. 4 to 20 or 8 to 20) amino acids in length containing *Asx or *Glx flanked by amino acid residues of perlecan, biglycan, decorin, fibrillin-1, or protocadherin.

[0083] The peptides described maybe used in assays providing therapeutic information for treating a pathological condition in a mammal.

[0084] The invention includes a method of immunoassay in which a biological sample is contacted with an immunological binding agent in the presence of a peptide, as described, acting as a competition agent for binding to said immunological binding agent.

[0085] In a further aspect there is provided a test kit comprising (a) an immunological binding partner as described herein or (b) a peptide as described herein. Versions of the test kit combine (a) and (b), optionally in combination with one or more apparatus in which to perform an immunoassay, an antibody-enzyme conjugate, a substrate for an enzyme component of an antibody-enzyme conjugate, an enzyme-substrate reaction stopping composition, or a wash solution, a carrier bound to said binding partner or a detectable label bound to said binding partner.

[0086] The invention also includes cell lines (e.g. hybridomas) that produce monoclonal antibodies immunoreactive with the above-mentioned proteins, fragments thereof or synthetic peptides. The invention further includes monoclonal antibodies produced by the fused cell hybrids, and those antibodies (as well as binding fragments thereof, e.g. Fab) coupled to a detectable marker. Examples of detectable markers include, but are not limited to, enzymes, chromophores, fluorophores, co-enzymes, enzyme inhibitors, chemiluminescent materials, paramagnetic metals, spin labels and radioisotopes.

[0087] The methods of the invention typically involve quantitating in a biological fluid the concentration of particular isopeptide fragments derived from tissue degradation. In a representative assay, isopeptide fragments in the biological fluid and a synthetic peptide immobilized on a solid support are contacted with an immunological binding partner, which is immunoreactive with the synthetic peptide as well as the isopeptide fragments, thereby generating a competition assay.

[0088] The biological fluid may be used as it is, or it may be purified prior to the contacting step. This purification step may be accomplished using a number of standard procedures, including but not limited to, cartridge adsorption and elution, molecular sieve chromatography, dialysis, ion exchange, alumina chromatography, hydroxyapatite chromatography, and combinations thereof.

[0089] The invention also includes test kits useful for quantifying in a body fluid (or cell supernatant) the amount of isopeptide fragments derived from the degradation of tissue. The kits comprise at least one immunological binding partner, e.g. a monoclonal or polyclonal antibody specific for a peptide derived from the degradation of a relevant protein. If desired, the immunological binding partner of the test kit may be coupled to detectable markers such as the ones described above. Generally speaking, the immunological binding partner is therefore also useful as a diagnostic agent.

[0090] Various non-immunological methods of assay may also be employed for analysis and identification of isoaspartyl, D-aspartyl and D-isoaspartyl within proteins or peptides.

[0091] The presence isoapartyl (D-aspartyl and D-isoaspartyl) within a peptide or protein may be determined by methods well known in the art. These methods include the use of 1) chromatographic methods, 2) electrophoretic techniques, 3) enzymatic methods 4) immunological methods, 5) chemical methods or combinations thereof. A review of current methods for detection and quantitation of isoaspartate is provided in by Aswad and Guzzetta (Aswad and Guzzetta, 1995).

[0092] Chromatographic methods maybe used, such as reversed phase HPLC (Cloos and Fledelius 2000) and hydrophobic interaction chromatography (Di Donato et al 1993). The structural alterations associated with isoaspartyl (D-aspartyl and D-isoaspartyl) formation can produce significant changes in the retention of peptides in various chromatographic methods.

[0093] Electrophoretic techniques as native gel-electrophoresis (Potter et al 1993), capillary electrophoresis (Stevenson et al 1993) and thin-layer electrophoresis (Aswad et at 1987) can be used to separate and may be used as means to identify such changes.

[0094] Enzymatic methods, including the use of the IAMT enzyme (EC 2.1.1.77) or of specific proteases specifically cleaving at the N or C of isoaspartyl residues may be used to identify such changes.

[0095] The enzyme IsoAspartyl (D-aspartyl) MethylTransferase (IAMT), EC 2.1.1.77 provides a method for selectively labelling isoaspartyl (D-aspartyl) sites with-methyl groups. This enzyme has the ability to recognise and methylate atypical isoAsp and D-Asp residues. By employing a radioactively labelled methyl-donor (³H or 14C-methyl), isomerised (or optically inverted) proteins or peptides incubated with this enzyme will be radioactively labelled, and labelling of the protein can be detected by measuring the incorporated radioactivity. A kit (IsoQuant™), based on this principle is commercially available.

[0096] Alternative methods for IAMT-dependent isoAsp analysis have recently been described. Thus isoAsp formation has been detected utilizing HPLC in conjunction with UV-detection to monitor production of S-adenosyl-L-Homocysteine (Aswad et al 2000).

[0097] Proteases that selectively cleave at the N or C of isoaspartyl (D-aspartyl or D-isoaspartyl) may be useful for isoAsp analysis. As an example of this the Carboxypeptidase Y recognises L-isoaspartyl sites as if they were C-terminal amino acids and thereby acts as an “endoproteinase iso-asp-N” (Johnson and Aswad 1990). This action combined with normal CP-Y sequential clipping at the real terminus, can result in the production isoaspartyl dipeptides that can be charactized by amino acid analysis techniques. Such an approach may be useful in tracking down sites of isoAspartyl formation in proteins or peptides.

[0098] The Edman degradation is an important method for determining the location of an Asx alteration in a peptide, especially when used in conjunction with a complementary technique such as mass spectrometry or enzymatic methylation of isoaspartate. It has long been known that the presence of an isopeptides bond causes blockage of the Edman degradation because the extra carbon(s) in the backbone of isopeptides prevent cyclization of the phenylthiocarbamyl peptide to form the anilinothiazolone derivative (Smyth et al 1963). Thus a sequencing block at Asx (or Glx) sites provides powerful, albeit indirect, evidence for the presence of an isopeptide bond.

[0099] Matsuo and Narita have described a method based on Oxasolone Formation for selectively labelling the C-terminal α-carboxylamino acid of a peptide (Matsuo and Narita 1975). This method can be used to label internal isoaspartyl residues since they contain a free α-carboxyl. As an example of this Di Donato and colleagues have used 3H-labelling to confirm the presence of isoaspartate in peptides derived from bovine seminal ribonuclease and pancreatic ribonuclease.

[0100] Alkaline hydroxylamine has been used to cleave proteins mainly at Asn-Gly bonds The relative selectivity of this method is due to the propensity of Asn-Gly to be rapidly deamidated via the succinimide pathway at alkaline pH.

[0101] The invention will be further described and illustrated by the following examples, in which reference is made to the accompanying drawings, in which:

[0102]FIG. 1 shows in panels A and B the results of size exclusion chromatography of trypsin digested extracted (soluble fraction) and non-extractable (pellet) proteins from cartilage as described in Example 1.

[0103]FIG. 2 shows the results of reverse phase HPLC fractionation of low molecular weight fraction from tryptic digest of Gnd-HCl pellet, with typing of isoAsp/DAsp sites, as described in Example 1.

[0104]FIG. 3 shows results from the competitive β-COMP assay of example 3. Peptide standard curves with 20 μl/well of the isomerised (β) form and the non-isomerised (α) form of COMP peptide. 50% inhibition of the signal (Bo) was obtained with 38 ng/ml β-COMP peptide and 435 ng/ml α-COMP peptide.

[0105]FIG. 4 shows the results of β-COMP measurements in serum from 26 healthy individuals and 36 Osteo-arthritis patients. The samples were measured in the β-COMP ELISA (example 3). The significance of the difference between the groups was calculated by non-parametric T-Test to p=0.0015.

EXAMPLE 1 Purification of Isomerised and/or Optically Inverted Protein Fragments Derived from Cartilage, and Identification of Specific Isomerisation and/or Optical Inversion Sites in Cartilage Proteins

[0106]

[0107] Cartilage samples from two deceased human subjects (each approximately 20 mg wet-weight) (obtained from the department of forensic pathology of Copenhagen university hospital), were homogenized (with an ultra-turax homogeniser) and treated with 4 M guanidiniumhydrochloride (Gnd-HCl) for 48 h at 4° C. The extraction mixture was subsequently centrifuged at 27,000×g and the supernatant and pellet separated. Gnd-HCl was removed by extensive dialysis against Milli-Q water (MWCO=10 kDa, spectropor dialysis membranes) and lyophilised. Aliquots of the “Pellet” and “supernatants” were subsequently digested with trypsin (enzyme:substrate 1:25). Tryptic digests (of Gnd-HCl pellet and Gnd-HCl supernatant from extraction of cartilage) were subjected to size-exclusion chromatography (SEC) on a 500 ml Sephadex G-75 column. Eluents were collected in fractions and measured in relevant assays (IAMT, CartiLaps and AG1-ELISA). The eluate from the size exclusion column was analysed by OD214 nm, and after lyophilization and re-solubilization in phosphate buffered saline (PBS) in an enzyme assay using IAMT for detection of isomerised aspartate residues. IAMT-reactivity was found primarily in the medium and low-molecular weight fractions. No CartiLaps activity was found in eluents, and AG1-1 activity was almost exclusively restricted to the medium molecular weight fractions. Results are shown in FIG. 1.

[0108] Low molecular fractions were pooled, lyophilised and further separated by reversed phase HPLC. Low molecular fractions from SEC of tryptic digests (of Gnd-HCl supernatant and Gnd-HCl pellet, FIG. 1) were separated by R.P. HPLC. Eluents were collected in fractions and measured in the IAMT assay. Several IAMT-reactive fractions were found, these were further purified by R.P.HPLC and subsequently subjected to amino acid sequencing and mass spectrometry to identify isoAsp (D-Asp)-containing sequences. Results as shown in FIG. 2.

[0109] IAMT-positive fractions from the first HPLC separation were isolated and subjected to a second R.P. HPLC run, eluents were collected in fractions and measured in the IAMT assay. Purified fragments were subjected to mass spectrometry and amino acid sequencing for identification of isomerisation/optical inversion sites. The following cartilage protein fragments were identified: 5 in Aggrecan, 1 in Decorin, 1 in Biglycan, 1 in perlecan, 1 in protocadherin, 1 in Fibrillin and 1 in collagen type II.

[0110] Aggrecan site 1 (Pool 3 Frac 7, 8): LYPNOTGLPDPLSR (SEQ ID NO 12)

[0111] Aggrecan site 2 (Pool 2 Frac 82): SAIIATEQLQAAYEDGFHQC (SEQ ID NO 13)

[0112] Aggrecan site 3 (Frac 3.88 pool 1): LATTGOLYLAWQAGMDM (SEQ ID NO 14)

[0113] Aggrecan site 4 (Pool 9 and 10, trial 105): TGEDFVDIPENFFGV (SEQ ID NO 15)

[0114] Aggrecan site 5 (Pool 5, Frac 28): VSLPNYPAIPSDATLEVQSLRSNDS GVYR (SEQ ID NO 17)

[0115] Decorin (Trial 100, Frac 112): IADTNITSIPQGLPPSLTELLDG (SEQ ID NO 4)

[0116] Perlecan (Frac 3.75, pool 2): YPVRIESSSASLANGHTL (SEQ ID NO 2)

[0117] Protocadherin gamma A11 (Frac 3.8, pool 2): YEOFRDLELR (SEQ ID NO 9)

[0118] Fibrillin-1 (Frac 3.8, pool 2): YEOFSGGCQDINE (SEQ ID NO 11)

[0119] Biglycan (Trial 91, Frac 4.36.2): FTLDDGPFMMNDE (SEQ ID NO 6)

[0120] Collagen type II (Trial 91, Frac 4.36.1): EGSXGADGPXGRDG (SEQ ID NO 18)

[0121] The amino acid residues given in bold denotes isomerisation and optical inversion prone Aspartate and asparagine residues. The underlined residues denote the amino acids identified by sequencing the rest are extrapolated from the fragment mass identified by mass spectroscopy.

EXAMPLE 2 Development of Antibodies Specific for Isomerised and/or Optically Inverted Proteins or Protein Fragments and Generation of Immunoassays Specific for Isomerised and/or Optically Inverted Proteins or Protein Fragments

[0122] Immunogen for generation of a specific antiserum can be generated by two procedures. The authentic protein or fragments of the protein from an extra-cellular tissue can be purified by conventional chromatographic procedures as outlined in example 1. The isomerisation degree of the purified protein or protein fragments can be determined by use of the IAMT assay, and thus proteins or protein fragments containing a high isomerisation degree (i.e. more than 0.5 mol isoAsp/mol protein or protein fragment) can be used for the immunizations. Alternatively synthetic peptides representing the isomerisation sites identified in a given extra-cellular matrix protein can be synthesized by conventional peptide synthesis methods (please refer to ‘Stewart, J. Young, J., “Solid phase peptide synthesis” Pierce Chemical Company, Rockford, Ill., USA 1984’ and ‘Atherton, E., Sheppard, R. C., “Solid phase peptide synthesis: a practical approach”, IRL Press, Oxford, 1989’). Synthetic peptide can be made containing an isomerised and/or racemised Asx or Glx residue and a suitable number of amino acid residues on both sides of the residue (i.e. 1-15 amino acid residues, preferably 2-6) derived from the primary sequence of the given isomerisation prone extra-cellular matrix protein.

[0123] A synthetic peptide or protein or protein fragment (‘β-antigen’) containing an isomerised and/or racemised Asx or Glx residue can be used for immunisation of rabbits and generation of a specific antiserum as described in the following. The β-antigen is conjugated to a carrier protein by the use of the covalent cross-linking agent 1-ethyl-3-[3-dimethylaminopropyl]carbodi-imide hydrocloride (CDI) (Pierce, Rockford, Ill., USA). A conjugate of the β-antigen peptide is prepared essentially according to: Greg T. Hermanson, ‘Bioconjugate techniques’ 1996, Academic press, San Diego, USA. Briefly described the CDI conjugates are prepared as follows: One-hundred mg of Thyroglobulin is dissolved in 10 ml to a concentration of 10 mg/ml in 0.05 M MES, 0.5 M NaCl, pH 6.0. One-hundred μl of the two following reagents (to a final concentration of 4 mM CDI, corresponding to approximately 100 fold molar excess of CDI to thyroglobulin, and 10 mM NHS) is added, and the solution is left to mix 15 min at room temperature (18-22° C.). CDI: 0.4 M CDI stock: 76.7 mg in 1 ml water prepared immediately prior to use. NHS: 1 M sulfo-NHS stock: 217.1 mg in 1 ml water prepared immediately prior to use.

[0124] Excess cross-linking reactants (CDI) is removed by gel-filtration on four NAP25 de-salting columns (Amersham Pharmacia Biotech, Uppsala, Sweden) into 10 mM Na-Phosphate pH 9.0. The de-salted activated thyroglobulin is pooled and divided into 6 portions of 2 ml. Immediately following the gel-filtration peptide solutions: 2 ml 4 mg/ml in 0.1 M Na-Phosphate pH 9.0 is added to each vial. A control conjugation is carried out with an irrelevant peptide. The coupling reaction is allowed to proceed for two hours at room temperature.

[0125] The β-antigen conjugates are changes into PBS (pH 7.4) by gel-filtration on Sephadex G25 columns (Amersham Pharmacia Biotech, Uppsala, Sweden), and the concentration is adjusted to 2 mg/ml in PBS.

[0126] Rabbits (strain SSC:CPH), are immunised sub-cutaneously with 1 ml 0.25 mg/ml of the vaccinein phosphate buffered saline (PBS), containing 50% Freunds incomplete adjuvant. Rabbits are boosted after initial immunisations at two weeks intervals. After the three first boosts, subsequent booster immunisations are performed at one month intervals. Pre-immune bleed is collected before immunisation and test bleeds are collected one week after the 2^(nd) immunisation to monitor serum antibody levels. Bleeds are subsequently collected one week after the 5^(th) and 6^(th) immunisation.

[0127] The specificity of the rabbit bleed are tested on a micro titre plates (MTP) coated with a β-antigen conjugate prepred with the same β-antigen as used for the immunization. The β-antigen is concugated to bovine serum albumin (BSA) by us of the bi-functional cross-linker bis[sulfosuccinimidyl] suberate (BS³). This crosslinking reagent is obtained from Pierce (Rockford, Ill., USA) and the conjugation is prepared essentially as disclosed by the manufacturer. Briefly described the following procedure. Peptide stocks (β-antigen) are prepared in freshly filtered PBS to 2 mg/ml. The carrier protein (BSA) is prepared in 3 mg/ml concentration in PBS (approximately 50 fold molar excess of peptide). 200 μl of carrier protein is mixed with 200 μl peptide solution in an Eppendorf tube. BS3 (Bis-(sulfosuccinimidyl) suberate) 6 mg/ml is prepared in 5 mM sodium-citrate pH 5.0 (25 fold molar excess of cross-linker compared to carrier protein). 50 μl of the cross-linker is added to the carrier and peptide solution, which is vortexed and placed on a mixer at room temperature for 30 min. 50 μl 0.25 M glycine pH 7.5 is added to the solutions. The incubation is continued for 15 min., Whereafter the conjugates are desalted on NAP5 column and protein concentrations are determined by the Bradford or Loewry methods. MTP are coated with 10 ng/ml of the BSA-BS³-β-antigen conjugate and used for screening of serum from the immunized rabbits. The rabbit antiserum is diluted in PBS containing 1% BSA and 0.1% Tween in a suitable dilution for obtaining an appropriate ELISA signal (a suitable signal is between 1 and 3 absorbency units and a suitable dilution will typically be in the range between 1000 fold dilution and 100,000 fold dilution). The bound antibody in this competitive assay format was detected by use of a secondary peroxidase conjugated goat anti rabbit antibody and a chromogenic peroxidase substrate.

EXAMPLE 3 Generation of Antibodies Specific for an Isomerised Fragment of Cartilage Oligomeric Matrix Protein (β-COMP) and Development of a β-COMP Specific Immunoassay

[0128] Peptide Conjugation

[0129] A synthetic peptide derived from human cartilage oligomeric matrix protein (COMP) containing an isomerised Asp residue within the sequence AQED_(β)SDH (‘β-COMP’) SEQ ID NO:19 was used for immunisation of mice and generation of a specific antiserum as described in the following. The β-COMP peptide was conjugated to a carrier protein by the use of the covalent cross-linking agent 1-ethyl-3-[3-dimethylaminopropyl]carbodi-imide hydrocloride (CDI) (Pierce, Rockford, Ill., USA). The β-COMP conjugate was prepared essentially according to: Greg T. Hermanson, ‘Bioconjugate techniques’ 1996, Academic press, San Diego, USA. Briefly described the CDI conjugates were prepared as follows: One-hundred mg of thyroglobulin was dissolved to a concentration of 10 mg/ml in 0.05 M MES, 0.5 M NaCl, pH 6.0 in a total volume of 10 ml. one-hundred μl containing a final concentration of 4 mM CDI and 10 mM NHS was added to the thyroglobulin solution. The solution was left to mix 15 min at room temperature (18-22° C.). CDI: 0.4 M CDI stock: 76.7 mg in 1 ml water prepared immediately prior to use. N-hydroxysulfosuccinimide (NHS from Pierce, Rockford, Ill., USA): 1 M NHS stock: 217.1 mg in 1 ml water prepared immediately prior to use.

[0130] Excess cross-linking reactants (CDI) were removed by gel-filtration on four NAP25 de-salting columns (Amersham Pharmacia Biotech, Uppsala, Sweden) into 10 mM Na-Phosphate pH 9.0. The de-salted activated thyroglobulin was pooled and divided into 6 portions of 2 ml. Immediately following the gel-filtration 2 ml β-COMP peptide solution of 4 mg/ml in 0.1 M Na-Phosphate pH 9.0 was added to each vial. A control conjugation was carried out with an irrelevant peptide. The coupling reaction was allowed to proceed for two hours at room temperature.

[0131] The β-COMP conjugates were changed into phosphate buffered saline (PBS) (pH 7.4) by gel-filtration on Sephadex G25 columns (Amersham Pharmacia Biotech, Uppsala, Sweden), and the concentration was adjusted to 0.5 mg/ml in PBS.

[0132] Immunisation

[0133] Mice (strain Balb/C-J) were immunised sub-cutaneously with 0.2 ml 0.25 mg/ml β-COMP conjugate in PBS, containing 50% Freunds incomplete adjuvant. Mice were boosted after initial immunisations at two weeks intervals. Pre-immune bleeds were collected before immunisation and test bleeds were collected one week after the 2^(nd) immunisation and after subsequent booster immunisations to monitor serum antibody levels.

[0134] Specificity

[0135] The specificity of the mouse bleeds were tested on strept-avidin micro titer plates (Micro-coat, Munich, Germany) coated with 10 ng/ml biotinylated-β-COMP. The β-COMP biotinylation was prepared using standard conjugation techniques with a succinimide activated form of biotin, according to the manufactures instructions (Pierce, Rockford, USA). The mouse sera were diluted in PBS containing 1% BSA and 0.1% Tween (PBS-BTB) to a dilution giving an appropriate ELISA signal (OD-value of ≈2). Specificity for the isomerised form of the COMP peptide was tested by competion with both the isomerised (β) and the non-isomersied (α) form of the COMP peptide. The mouse serum that showed the highest degree of specificity for β-COMP was used for development of a competitive assay for measurement of β-COMP in serum samples (FIG. 3).

[0136] Briefly the assay was set-up as follows; streptavidin precoated micro titer plates (MicroCoat, Munich, Germany) were incubated with 100 μl/well of 0.5 ng/ml biotinylated-β-COMP in PBS-BTB at 20° C. for 30 minutes at a shaking table. The plates were washed 5 times in PBS with 0.1% Tween 20 (PBS-T), followed by overnight incubation at 4° C. with 100 μl/well mouse-anti-β-COMP sera diluted 1:8000 in PBS-BTB and 20 μl/well of standards containing 0.5, 1.5, 4.5, 13.5, 41.5, 125 and 375 ng/ml β-COMP peptide in PBS-BTB or 20 μl/well of serum samples from patients with osteo-arthritis or healthy individuals. The plates were washed five times in PBS-T. 100 μl secondary peroxidase conjugated sheep-anti-mouse IgG antibody (Jackson Immuno Research, Pennsylvania, USA) diluted 1:2000 in PBS-BTB was added to each well and incubated for 60 minutes at 20° C. on a shaking table. After washing five times in PBS-T the amount of bound peroxidase conjugated antibody was detected by 15 minutes incubation with 100 μl/well of a chromogenic peroxidase substrate (3,3′,5,5′-tetramethyl-benzidine (TMB) from KemEnTek, Copenhagen, Denmark) on a shaking table. The reaction was stopped with 100 μl/well of 0.18 M H₂SO₄ and micro titer plates were read at 450/650 nm.

[0137] The amount of β-COMP peptide in serum samples from healthy individuals, patients with osteo-arthritis (OA) (FIG. 4) and rheumatoid arthritis (RA) (not shown) were calculated from the β-COMP peptide standard curve.

CONCLUSION

[0138] It is apparent that circulating levels of β-COMP fragments can be measured in normal adult individuals as well as in patients with arthritis. In the OA and RA patients the levels of β-COMP is significantly elevated compared to the healthy controls. This demonstrates that the β-COMP marker measured in a specific immunoassay employing a polyclonal antiserum specific for the human COMP derived sequence AQED_(β)SDH reflects the elevated cartilage turnover associated with the pathologic joint tissue processes in RA and OA.

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[0182] 44. Smyth D G, Stein W H and Moore S (1963) J Biol Chem 238: 227-234.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 20 <210> SEQ ID NO 1 <211> LENGTH: 4391 <212> TYPE: PRT <213> ORGANISM: Perlecan Homo sapiens <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: AAA52700 <309> DATABASE ENTRY DATE: 1994-11-08 <400> SEQUENCE: 1 Met Gly Trp Arg Ala Pro Gly Ala Leu Leu Leu Ala Leu Leu Leu His 1 5 10 15 Gly Arg Leu Leu Ala Val Thr His Gly Leu Arg Ala Tyr Asp Gly Leu 20 25 30 Ser Leu Pro Glu Asp Ile Glu Thr Val Thr Ala Ser Gln Met Arg Trp 35 40 45 Thr His Ser Tyr Leu Ser Asp Asp Glu Tyr Met Leu Ala Asp Ser Ile 50 55 60 Ser Gly Asp Asp Leu Gly Ser Gly Asp Leu Gly Ser Gly Asp Phe Gln 65 70 75 80 Met Val Tyr Phe Arg Ala Leu Val Asn Phe Thr Arg Ser Ile Glu Tyr 85 90 95 Ser Pro Gln Leu Glu Asp Ala Gly Ser Arg Glu Phe Arg Glu Val Ser 100 105 110 Glu Ala Val Val Asp Thr Leu Glu Ser Glu Tyr Leu Lys Ile Pro Gly 115 120 125 Asp Gln Val Val Ser Val Val Phe Ile Lys Glu Leu Asp Gly Trp Val 130 135 140 Phe Val Glu Leu Asp Val Gly Ser Glu Gly Asn Ala Asp Gly Ala Gln 145 150 155 160 Ile Gln Glu Met Leu Leu Arg Val Ile Ser Ser Gly Ser Val Ala Ser 165 170 175 Tyr Val Thr Ser Pro Gln Gly Phe Gln Phe Arg Arg Leu Gly Thr Val 180 185 190 Pro Gln Phe Pro Arg Ala Cys Thr Glu Ala Glu Phe Ala Cys His Ser 195 200 205 Tyr Asn Glu Cys Val Ala Leu Glu Tyr Arg Cys Asp Arg Arg Pro Asp 210 215 220 Cys Arg Asp Met Ser Asp Glu Leu Asn Cys Glu Glu Pro Val Leu Gly 225 230 235 240 Ile Ser Pro Thr Phe Ser Leu Leu Val Glu Thr Thr Ser Leu Pro Pro 245 250 255 Arg Pro Glu Thr Thr Ile Met Arg Gln Pro Pro Val Thr His Ala Pro 260 265 270 Gln Pro Leu Leu Pro Gly Ser Val Arg Pro Leu Pro Cys Gly Pro Gln 275 280 285 Glu Ala Ala Cys Arg Asn Gly His Cys Ile Pro Arg Asp Tyr Leu Cys 290 295 300 Asp Gly Gln Glu Asp Cys Glu Asp Gly Ser Asp Glu Leu Asp Cys Gly 305 310 315 320 Pro Pro Pro Pro Cys Glu Pro Asn Glu Phe Pro Cys Gly Asn Gly His 325 330 335 Cys Ala Leu Lys Leu Trp Arg Cys Asp Gly Asp Phe Asp Cys Glu Asp 340 345 350 Arg Thr Asp Glu Ala Asn Cys Pro Thr Lys Arg Pro Glu Glu Val Cys 355 360 365 Gly Pro Thr Gln Phe Arg Cys Val Ser Thr Asn Met Cys Ile Pro Ala 370 375 380 Ser Phe His Cys Asp Glu Glu Ser Asp Cys Pro Asp Arg Ser Asp Glu 385 390 395 400 Phe Gly Cys Met Pro Pro Gln Val Val Thr Pro Pro Arg Glu Ser Ile 405 410 415 Gln Ala Ser Arg Gly Gln Thr Val Thr Phe Thr Cys Val Ala Ile Gly 420 425 430 Val Pro Thr Pro Ile Ile Asn Trp Arg Leu Asn Trp Gly His Ile Pro 435 440 445 Ser His Pro Arg Val Thr Val Thr Ser Glu Gly Gly Arg Gly Thr Leu 450 455 460 Ile Ile Arg Asp Val Lys Glu Ser Asp Gln Gly Ala Tyr Thr Cys Glu 465 470 475 480 Ala Met Asn Ala Arg Gly Met Val Phe Gly Ile Pro Asp Gly Val Leu 485 490 495 Glu Leu Val Pro Gln Arg Gly Pro Cys Pro Asp Gly His Phe Tyr Leu 500 505 510 Glu His Ser Ala Ala Cys Leu Pro Cys Phe Cys Phe Gly Ile Thr Ser 515 520 525 Val Cys Gln Ser Thr Arg Arg Phe Arg Asp Gln Ile Arg Leu Arg Phe 530 535 540 Asp Gln Pro Asp Asp Phe Lys Gly Val Asn Val Thr Met Pro Ala Gln 545 550 555 560 Pro Gly Thr Pro Pro Leu Ser Ser Thr Gln Leu Gln Ile Asp Pro Ser 565 570 575 Leu His Glu Phe Gln Leu Val Asp Leu Ser Arg Arg Phe Leu Val His 580 585 590 Asp Ser Phe Trp Ala Leu Pro Glu Gln Phe Leu Gly Asn Lys Val Asp 595 600 605 Ser Tyr Gly Gly Ser Leu Arg Tyr Asn Val Arg Tyr Glu Leu Ala Arg 610 615 620 Gly Met Leu Glu Pro Val Gln Arg Pro Asp Val Val Leu Val Gly Ala 625 630 635 640 Gly Tyr Arg Leu Leu Ser Arg Gly His Thr Pro Thr Gln Pro Gly Ala 645 650 655 Leu Asn Gln Arg Gln Val Gln Phe Ser Glu Glu His Trp Val His Glu 660 665 670 Ser Gly Arg Pro Val Gln Arg Ala Glu Leu Leu Gln Val Leu Gln Ser 675 680 685 Leu Glu Ala Val Leu Ile Gln Thr Val Tyr Asn Thr Lys Met Ala Ser 690 695 700 Val Gly Leu Ser Asp Ile Ala Met Asp Thr Thr Val Thr His Ala Thr 705 710 715 720 Ser His Gly Arg Ala His Ser Val Glu Glu Cys Arg Cys Pro Ile Gly 725 730 735 Tyr Ser Gly Leu Ser Cys Glu Ser Cys Asp Ala His Phe Thr Arg Val 740 745 750 Pro Gly Gly Pro Tyr Leu Gly Thr Cys Ser Gly Cys Ser Cys Asn Gly 755 760 765 His Ala Ser Ser Cys Asp Pro Val Tyr Gly His Cys Leu Asn Cys Gln 770 775 780 His Asn Thr Glu Gly Pro Gln Cys Asn Lys Cys Lys Ala Gly Phe Phe 785 790 795 800 Gly Asp Ala Met Lys Ala Thr Ala Thr Ser Cys Arg Pro Cys Pro Cys 805 810 815 Pro Tyr Ile Asp Ala Ser Arg Arg Phe Ser Asp Thr Cys Phe Leu Asp 820 825 830 Thr Asp Gly Gln Ala Thr Cys Asp Ala Cys Ala Pro Gly Tyr Thr Gly 835 840 845 Arg Arg Cys Glu Ser Cys Ala Pro Gly Tyr Glu Gly Asn Pro Ile Gln 850 855 860 Pro Gly Gly Lys Cys Arg Pro Val Asn Gln Glu Ile Val Arg Cys Asp 865 870 875 880 Glu Arg Gly Ser Met Gly Thr Ser Gly Glu Ala Cys Arg Cys Lys Asn 885 890 895 Asn Val Val Gly Arg Leu Cys Asn Glu Cys Ala Asp Gly Ser Phe His 900 905 910 Leu Ser Thr Arg Asn Pro Asp Gly Cys Leu Lys Cys Phe Cys Met Gly 915 920 925 Val Ser Arg His Cys Thr Ser Ser Ser Trp Ser Arg Ala Gln Leu His 930 935 940 Gly Ala Ser Glu Glu Pro Gly His Phe Ser Leu Thr Asn Ala Ala Ser 945 950 955 960 Thr His Thr Thr Asn Glu Gly Ile Phe Ser Pro Thr Pro Gly Glu Leu 965 970 975 Gly Phe Ser Ser Phe His Arg Leu Leu Ser Gly Pro Tyr Phe Trp Ser 980 985 990 Leu Pro Ser Arg Phe Leu Gly Asp Lys Val Thr Ser Tyr Gly Gly Glu 995 1000 1005 Leu Arg Phe Thr Val Thr Gln Arg Ser Gln Pro Gly Ser Thr Pro 1010 1015 1020 Leu His Gly Gln Pro Leu Val Val Leu Gln Gly Asn Asn Ile Ile 1025 1030 1035 Leu Glu His His Val Ala Gln Glu Pro Ser Pro Gly Gln Pro Ser 1040 1045 1050 Thr Phe Ile Val Pro Phe Arg Glu Gln Ala Trp Gln Arg Pro Asp 1055 1060 1065 Gly Gln Pro Ala Thr Arg Glu His Leu Leu Met Ala Leu Ala Gly 1070 1075 1080 Ile Asp Thr Leu Leu Ile Arg Ala Ser Tyr Ala Gln Gln Pro Ala 1085 1090 1095 Glu Ser Arg Val Ser Gly Ile Ser Met Asp Val Ala Val Pro Glu 1100 1105 1110 Glu Thr Gly Gln Asp Pro Ala Leu Glu Val Glu Gln Cys Ser Cys 1115 1120 1125 Pro Pro Gly Tyr Arg Gly Pro Ser Cys Gln Asp Cys Asp Thr Gly 1130 1135 1140 Tyr Thr Arg Thr Pro Ser Gly Leu Tyr Leu Gly Thr Cys Glu Arg 1145 1150 1155 Cys Ser Cys His Gly His Ser Glu Ala Cys Glu Pro Glu Thr Gly 1160 1165 1170 Ala Cys Gln Gly Cys Gln His His Thr Glu Gly Pro Arg Cys Glu 1175 1180 1185 Gln Cys Gln Pro Gly Tyr Tyr Gly Asp Ala Gln Arg Gly Thr Pro 1190 1195 1200 Gln Asp Cys Gln Leu Cys Pro Cys Tyr Gly Asp Pro Ala Ala Gly 1205 1210 1215 Gln Ala Ala His Thr Cys Phe Leu Asp Thr Asp Gly His Pro Thr 1220 1225 1230 Cys Asp Ala Cys Ser Pro Gly His Ser Gly Arg His Cys Glu Arg 1235 1240 1245 Cys Ala Pro Gly Tyr Tyr Gly Asn Pro Ser Gln Gly Gln Pro Cys 1250 1255 1260 Gln Arg Asp Ser Gln Val Pro Gly Pro Ile Gly Cys Asn Cys Asp 1265 1270 1275 Pro Gln Gly Ser Val Ser Ser Gln Cys Asp Ala Ala Gly Gln Cys 1280 1285 1290 Gln Cys Lys Ala Gln Val Glu Gly Leu Thr Cys Ser His Cys Arg 1295 1300 1305 Pro His His Phe His Leu Ser Ala Ser Asn Pro Asp Gly Cys Leu 1310 1315 1320 Pro Cys Phe Cys Met Gly Ile Thr Gln Gln Cys Ala Ser Ser Ala 1325 1330 1335 Tyr Thr Arg His Leu Ile Ser Thr His Phe Ala Pro Gly Asp Phe 1340 1345 1350 Gln Gly Phe Ala Leu Val Asn Pro Gln Arg Asn Ser Arg Leu Thr 1355 1360 1365 Gly Glu Phe Thr Val Glu Pro Val Pro Glu Gly Ala Gln Leu Ser 1370 1375 1380 Phe Gly Asn Phe Ala Gln Leu Gly His Glu Ser Phe Tyr Trp Gln 1385 1390 1395 Leu Pro Glu Thr Tyr Gln Gly Asp Lys Val Ala Ala Tyr Gly Gly 1400 1405 1410 Lys Leu Arg Tyr Thr Leu Ser Tyr Thr Ala Gly Pro Gln Gly Ser 1415 1420 1425 Pro Leu Ser Asp Pro Asp Val Gln Ile Thr Gly Asn Asn Ile Met 1430 1435 1440 Leu Val Ala Ser Gln Pro Ala Leu Gln Gly Pro Glu Arg Arg Ser 1445 1450 1455 Tyr Glu Ile Met Phe Arg Glu Glu Phe Trp Arg Arg Pro Asp Gly 1460 1465 1470 Gln Pro Ala Thr Arg Glu His Leu Leu Met Ala Leu Ala Asp Leu 1475 1480 1485 Asp Glu Leu Leu Ile Arg Ala Thr Phe Ser Ser Val Pro Leu Val 1490 1495 1500 Ala Ser Ile Ser Ala Val Ser Leu Glu Val Ala Gln Pro Gly Pro 1505 1510 1515 Ser Asn Arg Pro Arg Ala Leu Glu Val Glu Glu Cys Arg Cys Pro 1520 1525 1530 Pro Gly Tyr Ile Gly Leu Ser Cys Gln Asp Cys Ala Pro Gly Tyr 1535 1540 1545 Thr Arg Thr Gly Ser Gly Leu Tyr Leu Gly His Cys Glu Leu Cys 1550 1555 1560 Glu Cys Asn Gly His Ser Asp Leu Cys His Pro Glu Thr Gly Ala 1565 1570 1575 Cys Ser Gln Cys Gln His Asn Ala Ala Gly Glu Phe Cys Glu Leu 1580 1585 1590 Cys Ala Pro Gly Tyr Tyr Gly Asp Ala Thr Ala Gly Thr Pro Glu 1595 1600 1605 Asp Cys Gln Pro Cys Ala Cys Pro Leu Thr Asn Pro Glu Asn Met 1610 1615 1620 Phe Ser Arg Thr Cys Glu Ser Leu Gly Ala Gly Gly Tyr Arg Cys 1625 1630 1635 Thr Ala Cys Glu Pro Gly Tyr Thr Gly Gln Tyr Cys Glu Gln Cys 1640 1645 1650 Gly Pro Gly Tyr Val Gly Asn Pro Ser Val Gln Gly Gly Gln Cys 1655 1660 1665 Leu Pro Glu Thr Asn Gln Ala Pro Leu Val Val Glu Val His Pro 1670 1675 1680 Ala Arg Ser Ile Val Pro Gln Gly Gly Ser His Ser Leu Arg Cys 1685 1690 1695 Gln Val Ser Gly Ser Pro Pro His Tyr Phe Tyr Trp Ser Arg Glu 1700 1705 1710 Asp Gly Arg Pro Val Pro Ser Gly Thr Gln Gln Arg His Gln Gly 1715 1720 1725 Ser Glu Leu His Phe Pro Ser Val Gln Pro Ser Asp Ala Gly Val 1730 1735 1740 Tyr Ile Cys Thr Cys Arg Asn Leu His Gln Ser Asn Thr Ser Arg 1745 1750 1755 Ala Glu Leu Leu Val Thr Glu Ala Pro Ser Lys Pro Ile Thr Val 1760 1765 1770 Thr Val Glu Glu Gln Arg Ser Gln Ser Val Arg Pro Gly Ala Asp 1775 1780 1785 Val Thr Phe Ile Cys Thr Ala Lys Ser Lys Ser Pro Ala Tyr Thr 1790 1795 1800 Leu Val Trp Thr Arg Leu His Asn Gly Lys Leu Pro Thr Arg Ala 1805 1810 1815 Met Asp Phe Asn Gly Ile Leu Thr Ile Arg Asn Val Gln Leu Ser 1820 1825 1830 Asp Ala Gly Thr Tyr Val Cys Thr Gly Ser Asn Met Phe Ala Met 1835 1840 1845 Asp Gln Gly Thr Ala Thr Leu His Val Gln Ala Ser Gly Thr Leu 1850 1855 1860 Ser Ala Pro Val Val Ser Ile His Pro Pro Gln Leu Thr Val Gln 1865 1870 1875 Pro Gly Gln Leu Ala Glu Phe Arg Cys Ser Ala Thr Gly Ser Pro 1880 1885 1890 Thr Pro Thr Leu Glu Trp Thr Gly Gly Pro Gly Gly Gln Leu Pro 1895 1900 1905 Ala Lys Ala Gln Ile His Gly Gly Ile Leu Arg Leu Pro Ala Val 1910 1915 1920 Glu Pro Thr Asp Gln Ala Gln Tyr Leu Cys Arg Ala His Ser Ser 1925 1930 1935 Ala Gly Gln Gln Val Ala Arg Ala Val Leu His Val His Gly Gly 1940 1945 1950 Gly Gly Pro Arg Val Gln Val Ser Pro Glu Arg Thr Gln Val His 1955 1960 1965 Ala Gly Arg Thr Val Arg Leu Tyr Cys Arg Ala Ala Gly Val Pro 1970 1975 1980 Ser Ala Thr Ile Thr Trp Arg Lys Glu Gly Gly Ser Leu Pro Pro 1985 1990 1995 Gln Ala Arg Ser Glu Arg Thr Asp Ile Ala Thr Leu Leu Ile Pro 2000 2005 2010 Ala Ile Thr Thr Ala Asp Ala Gly Phe Tyr Leu Cys Val Ala Thr 2015 2020 2025 Ser Pro Ala Gly Thr Ala Gln Ala Arg Met Gln Val Val Val Leu 2030 2035 2040 Ser Ala Ser Asp Ala Ser Pro Pro Gly Val Lys Ile Glu Ser Ser 2045 2050 2055 Ser Pro Ser Val Thr Glu Gly Gln Thr Leu Asp Leu Asn Cys Val 2060 2065 2070 Val Ala Gly Ser Ala His Ala Gln Val Thr Trp Tyr Arg Arg Gly 2075 2080 2085 Gly Ser Leu Pro Pro His Thr Gln Val His Gly Ser Arg Leu Arg 2090 2095 2100 Leu Pro Gln Val Ser Pro Ala Asp Ser Gly Glu Tyr Val Cys Arg 2105 2110 2115 Val Glu Asn Gly Ser Gly Pro Lys Glu Ala Ser Ile Thr Val Ser 2120 2125 2130 Val Leu His Gly Thr His Ser Gly Pro Ser Tyr Thr Pro Val Pro 2135 2140 2145 Gly Ser Thr Arg Pro Ile Arg Ile Glu Pro Ser Ser Ser His Val 2150 2155 2160 Ala Glu Gly Gln Thr Leu Asp Leu Asn Cys Val Val Pro Gly Gln 2165 2170 2175 Ala His Ala Gln Val Thr Trp His Lys Arg Gly Gly Ser Leu Pro 2180 2185 2190 Ala Arg His Gln Thr His Gly Ser Leu Leu Arg Leu His Gln Val 2195 2200 2205 Thr Pro Ala Asp Ser Gly Glu Tyr Val Cys His Val Val Gly Thr 2210 2215 2220 Ser Gly Pro Leu Glu Ala Ser Val Leu Val Thr Ile Glu Ala Ser 2225 2230 2235 Val Ile Pro Gly Pro Ile Pro Pro Val Arg Ile Glu Ser Ser Ser 2240 2245 2250 Ser Thr Val Ala Glu Gly Gln Thr Leu Asp Leu Ser Cys Val Val 2255 2260 2265 Ala Gly Gln Ala His Ala Gln Val Thr Trp Tyr Lys Arg Gly Gly 2270 2275 2280 Ser Leu Pro Ala Arg His Gln Val Arg Gly Ser Arg Leu Tyr Ile 2285 2290 2295 Phe Gln Ala Ser Pro Ala Asp Ala Gly Gln Tyr Val Cys Arg Ala 2300 2305 2310 Ser Asn Gly Met Glu Ala Ser Ile Thr Val Thr Val Thr Gly Thr 2315 2320 2325 Gln Gly Ala Asn Leu Ala Tyr Pro Ala Gly Ser Thr Gln Pro Ile 2330 2335 2340 Arg Ile Glu Pro Ser Ser Ser Gln Val Ala Glu Gly Gln Thr Leu 2345 2350 2355 Asp Leu Asn Cys Val Val Pro Gly Gln Ser His Ala Gln Val Thr 2360 2365 2370 Trp His Lys Arg Gly Gly Ser Leu Pro Val Arg His Gln Thr His 2375 2380 2385 Gly Ser Leu Leu Arg Leu Tyr Gln Ala Ser Pro Ala Asp Ser Gly 2390 2395 2400 Glu Tyr Val Cys Arg Val Leu Gly Ser Ser Val Pro Leu Glu Ala 2405 2410 2415 Ser Val Leu Val Thr Ile Glu Pro Ala Gly Ser Val Pro Ala Leu 2420 2425 2430 Gly Val Thr Pro Thr Val Arg Ile Glu Ser Ser Ser Ser Gln Val 2435 2440 2445 Ala Glu Gly Gln Thr Leu Asp Leu Asn Cys Leu Val Ala Gly Gln 2450 2455 2460 Ala His Ala Gln Val Thr Trp His Lys Arg Gly Gly Ser Leu Pro 2465 2470 2475 Ala Arg His Gln Val His Gly Ser Arg Leu Arg Leu Leu Gln Val 2480 2485 2490 Thr Pro Ala Asp Ser Gly Glu Tyr Val Cys Arg Val Val Gly Ser 2495 2500 2505 Ser Gly Thr Gln Glu Ala Ser Val Leu Val Thr Ile Gln Gln Arg 2510 2515 2520 Leu Ser Gly Ser His Ser Gln Gly Val Ala Tyr Pro Val Arg Ile 2525 2530 2535 Glu Ser Ser Ser Ala Ser Leu Ala Asn Gly His Thr Leu Asp Leu 2540 2545 2550 Asn Cys Leu Val Ala Ser Gln Ala Pro His Thr Ile Thr Trp Tyr 2555 2560 2565 Lys Arg Gly Gly Ser Leu Pro Ser Arg His Gln Ile Val Gly Ser 2570 2575 2580 Arg Leu Arg Ile Pro Gln Val Thr Pro Ala Asp Ser Gly Glu Tyr 2585 2590 2595 Val Cys His Val Ser Asn Gly Ala Gly Ser Arg Glu Thr Ser Leu 2600 2605 2610 Ile Val Thr Ile Gln Gly Ser Gly Ser Ser His Val Pro Ser Val 2615 2620 2625 Ser Pro Pro Ile Arg Ile Glu Ser Ser Ser Pro Thr Val Val Glu 2630 2635 2640 Gly Gln Thr Leu Asp Leu Asn Cys Val Val Ala Arg Gln Pro Gln 2645 2650 2655 Ala Ile Ile Thr Trp Tyr Lys Arg Gly Gly Ser Leu Pro Ser Arg 2660 2665 2670 His Gln Thr His Gly Ser His Leu Arg Leu His Gln Met Ser Val 2675 2680 2685 Ala Asp Ser Gly Glu Tyr Val Cys Arg Ala Asn Asn Asn Ile Asp 2690 2695 2700 Ala Leu Glu Ala Ser Ile Val Ile Ser Val Ser Pro Ser Ala Gly 2705 2710 2715 Ser Pro Ser Ala Pro Gly Ser Ser Met Pro Ile Arg Ile Glu Ser 2720 2725 2730 Ser Ser Ser His Val Ala Glu Gly Glu Thr Leu Asp Leu Asn Cys 2735 2740 2745 Val Val Pro Gly Gln Ala His Ala Gln Val Thr Trp His Lys Arg 2750 2755 2760 Gly Gly Ser Leu Pro Ser His His Gln Thr Arg Gly Ser Arg Leu 2765 2770 2775 Arg Leu His His Val Ser Pro Ala Asp Ser Gly Glu Tyr Val Cys 2780 2785 2790 Arg Val Met Gly Ser Ser Gly Pro Leu Glu Ala Ser Val Leu Val 2795 2800 2805 Thr Ile Glu Ala Ser Gly Ser Ser Ala Val His Val Pro Ala Pro 2810 2815 2820 Gly Gly Ala Pro Pro Ile Arg Ile Glu Pro Ser Ser Ser Arg Val 2825 2830 2835 Ala Glu Gly Gln Thr Leu Asp Leu Lys Cys Val Val Pro Gly Gln 2840 2845 2850 Ala His Ala Gln Val Thr Trp His Lys Arg Gly Gly Asn Leu Pro 2855 2860 2865 Ala Arg His Gln Val His Gly Pro Leu Leu Arg Leu Asn Gln Val 2870 2875 2880 Ser Pro Ala Asp Ser Gly Glu Tyr Ser Cys Gln Val Thr Gly Ser 2885 2890 2895 Ser Gly Thr Leu Glu Ala Ser Val Leu Val Thr Ile Glu Pro Ser 2900 2905 2910 Ser Pro Gly Pro Ile Pro Ala Pro Gly Leu Ala Gln Pro Ile Tyr 2915 2920 2925 Ile Glu Ala Ser Ser Ser His Val Thr Glu Gly Gln Thr Leu Asp 2930 2935 2940 Leu Asn Cys Val Val Pro Gly Gln Ala His Ala Gln Val Thr Trp 2945 2950 2955 Tyr Lys Arg Gly Gly Ser Leu Pro Ala Arg His Gln Thr His Gly 2960 2965 2970 Ser Gln Leu Arg Leu His Leu Val Ser Pro Ala Asp Ser Gly Glu 2975 2980 2985 Tyr Val Cys Arg Ala Ala Ser Gly Pro Gly Pro Glu Gln Glu Ala 2990 2995 3000 Ser Phe Thr Val Thr Val Pro Pro Ser Glu Gly Ser Ser Tyr Arg 3005 3010 3015 Leu Arg Ser Pro Val Ile Ser Ile Asp Pro Pro Ser Ser Thr Val 3020 3025 3030 Gln Gln Gly Gln Asp Ala Ser Phe Lys Cys Leu Ile His Asp Gly 3035 3040 3045 Ala Ala Pro Ile Ser Leu Glu Trp Lys Thr Arg Asn Gln Glu Leu 3050 3055 3060 Glu Asp Asn Val His Ile Ser Pro Asn Gly Ser Ile Ile Thr Ile 3065 3070 3075 Val Gly Thr Arg Pro Ser Asn His Gly Thr Tyr Arg Cys Val Ala 3080 3085 3090 Ser Asn Ala Tyr Gly Val Ala Gln Ser Val Val Asn Leu Ser Val 3095 3100 3105 His Gly Pro Pro Thr Val Ser Val Leu Pro Glu Gly Pro Val Trp 3110 3115 3120 Val Lys Val Gly Lys Ala Val Thr Leu Glu Cys Val Ser Ala Gly 3125 3130 3135 Glu Pro Arg Ser Ser Ala Arg Trp Thr Arg Ile Ser Ser Thr Pro 3140 3145 3150 Ala Lys Leu Glu Gln Arg Thr Tyr Gly Leu Met Asp Ser His Ala 3155 3160 3165 Val Leu Gln Ile Ser Ser Ala Lys Pro Ser Asp Ala Gly Thr Tyr 3170 3175 3180 Val Cys Leu Ala Gln Asn Ala Leu Gly Thr Ala Gln Lys Gln Val 3185 3190 3195 Glu Val Ile Val Asp Thr Gly Ala Met Ala Pro Gly Ala Pro Gln 3200 3205 3210 Val Gln Ala Glu Glu Ala Glu Leu Thr Val Glu Ala Gly His Thr 3215 3220 3225 Ala Thr Leu Arg Cys Ser Ala Thr Gly Ser Pro Ala Pro Thr Ile 3230 3235 3240 His Trp Ser Lys Leu Arg Ser Pro Leu Pro Trp Gln His Arg Leu 3245 3250 3255 Glu Gly Asp Thr Leu Ile Ile Pro Arg Val Ala Gln Gln Asp Ser 3260 3265 3270 Gly Gln Tyr Ile Cys Asn Ala Thr Ser Pro Ala Gly His Ala Glu 3275 3280 3285 Ala Thr Ile Ile Leu His Val Glu Ser Pro Pro Tyr Ala Thr Thr 3290 3295 3300 Val Pro Glu His Ala Ser Val Gln Ala Gly Glu Thr Val Gln Leu 3305 3310 3315 Gln Cys Leu Ala His Gly Thr Pro Pro Leu Thr Phe Gln Trp Ser 3320 3325 3330 Arg Val Gly Ser Ser Leu Pro Gly Arg Ala Thr Ala Arg Asn Glu 3335 3340 3345 Leu Leu His Phe Glu Arg Ala Ala Pro Glu Asp Ser Gly Arg Tyr 3350 3355 3360 Arg Cys Arg Val Thr Asn Lys Val Gly Ser Ala Glu Ala Phe Ala 3365 3370 3375 Gln Leu Leu Val Gln Gly Pro Pro Gly Ser Leu Pro Ala Thr Ser 3380 3385 3390 Ile Pro Ala Gly Ser Thr Pro Thr Val Gln Val Thr Pro Gln Leu 3395 3400 3405 Glu Thr Lys Ser Ile Gly Ala Ser Val Glu Phe His Cys Ala Val 3410 3415 3420 Pro Ser Asp Gln Gly Thr Gln Leu Arg Trp Phe Lys Glu Gly Gly 3425 3430 3435 Gln Leu Pro Pro Gly His Ser Val Gln Asp Gly Val Leu Arg Ile 3440 3445 3450 Gln Asn Leu Asp Gln Ser Cys Gln Gly Thr Tyr Ile Cys Gln Ala 3455 3460 3465 His Gly Pro Trp Gly Lys Ala Gln Ala Ser Ala Gln Leu Val Ile 3470 3475 3480 Gln Ala Leu Pro Ser Val Leu Ile Asn Ile Arg Thr Ser Val Gln 3485 3490 3495 Thr Val Val Val Gly His Ala Val Glu Phe Glu Cys Leu Ala Leu 3500 3505 3510 Gly Asp Pro Lys Pro Gln Val Thr Trp Ser Lys Val Gly Gly His 3515 3520 3525 Leu Arg Pro Gly Ile Val Gln Ser Gly Gly Val Val Arg Ile Ala 3530 3535 3540 His Val Glu Leu Ala Asp Ala Gly Gln Tyr Arg Cys Thr Ala Thr 3545 3550 3555 Asn Ala Ala Gly Thr Thr Gln Ser His Val Leu Leu Leu Val Gln 3560 3565 3570 Ala Leu Pro Gln Ile Ser Met Pro Gln Glu Val Arg Val Pro Ala 3575 3580 3585 Gly Ser Ala Ala Val Phe Pro Cys Ile Ala Ser Gly Tyr Pro Thr 3590 3595 3600 Pro Asp Ile Ser Trp Ser Lys Leu Asp Gly Ser Leu Pro Pro Asp 3605 3610 3615 Ser Arg Leu Glu Asn Asn Met Leu Met Leu Pro Ser Val Arg Pro 3620 3625 3630 Gln Asp Ala Gly Thr Tyr Val Cys Thr Ala Thr Asn Arg Gln Gly 3635 3640 3645 Lys Val Lys Ala Phe Ala His Leu Gln Val Pro Glu Arg Val Val 3650 3655 3660 Pro Tyr Phe Thr Gln Thr Pro Tyr Ser Phe Leu Pro Leu Pro Thr 3665 3670 3675 Ile Lys Asp Ala Tyr Arg Lys Phe Glu Ile Lys Ile Thr Phe Arg 3680 3685 3690 Pro Asp Ser Ala Asp Gly Met Leu Leu Tyr Asn Gly Gln Lys Arg 3695 3700 3705 Val Pro Gly Ser Pro Thr Asn Leu Ala Asn Arg Gln Pro Asp Phe 3710 3715 3720 Ile Ser Phe Gly Leu Val Gly Gly Arg Pro Glu Phe Arg Phe Asp 3725 3730 3735 Ala Gly Ser Gly Met Ala Thr Ile Arg His Pro Thr Pro Leu Ala 3740 3745 3750 Leu Gly His Phe His Thr Val Thr Leu Leu Arg Ser Leu Thr Gln 3755 3760 3765 Gly Ser Leu Ile Val Gly Asp Leu Ala Pro Val Asn Gly Thr Ser 3770 3775 3780 Gln Gly Lys Phe Gln Gly Leu Asp Leu Asn Glu Glu Leu Tyr Leu 3785 3790 3795 Gly Gly Tyr Pro Asp Tyr Gly Ala Ile Pro Lys Ala Gly Leu Ser 3800 3805 3810 Ser Gly Phe Ile Gly Cys Val Arg Glu Leu Arg Ile Gln Gly Glu 3815 3820 3825 Glu Ile Val Phe His Asp Leu Asn Leu Thr Ala His Gly Ile Ser 3830 3835 3840 His Cys Pro Thr Cys Arg Asp Arg Pro Cys Gln Asn Gly Gly Gln 3845 3850 3855 Cys His Asp Ser Glu Ser Ser Ser Tyr Val Cys Val Cys Pro Ala 3860 3865 3870 Gly Phe Thr Gly Ser Arg Cys Glu His Ser Gln Ala Leu His Cys 3875 3880 3885 His Pro Glu Ala Cys Gly Pro Asp Ala Thr Cys Val Asn Arg Pro 3890 3895 3900 Asp Gly Arg Gly Tyr Thr Cys Arg Cys His Leu Gly Arg Ser Gly 3905 3910 3915 Leu Arg Cys Glu Glu Gly Val Thr Val Thr Thr Pro Ser Leu Ser 3920 3925 3930 Gly Ala Gly Ser Tyr Leu Ala Leu Pro Ala Leu Thr Asn Thr His 3935 3940 3945 His Glu Leu Arg Leu Asp Val Glu Phe Lys Pro Leu Ala Pro Asp 3950 3955 3960 Gly Val Leu Leu Phe Ser Gly Gly Lys Ser Gly Pro Val Glu Asp 3965 3970 3975 Phe Val Ser Leu Ala Met Val Gly Gly His Leu Glu Phe Arg Tyr 3980 3985 3990 Glu Leu Gly Ser Gly Leu Ala Val Leu Arg Ser Ala Glu Pro Leu 3995 4000 4005 Ala Leu Gly Arg Trp His Arg Val Ser Ala Glu Arg Leu Asn Lys 4010 4015 4020 Asp Gly Ser Leu Arg Val Asn Gly Gly Arg Pro Val Leu Arg Ser 4025 4030 4035 Ser Pro Gly Lys Ser Gln Gly Leu Asn Leu His Thr Leu Leu Tyr 4040 4045 4050 Leu Gly Gly Val Glu Pro Ser Val Pro Leu Ser Pro Ala Thr Asn 4055 4060 4065 Met Ser Ala His Phe Arg Gly Cys Val Gly Glu Val Ser Val Asn 4070 4075 4080 Gly Lys Arg Leu Asp Leu Thr Tyr Ser Phe Leu Gly Ser Gln Gly 4085 4090 4095 Ile Gly Gln Cys Tyr Asp Ser Ser Pro Cys Glu Arg Gln Pro Cys 4100 4105 4110 Gln His Gly Ala Thr Cys Met Pro Ala Gly Glu Tyr Glu Phe Gln 4115 4120 4125 Cys Leu Cys Arg Asp Gly Phe Lys Gly Asp Leu Cys Glu His Glu 4130 4135 4140 Glu Asn Pro Cys Gln Leu Arg Glu Pro Cys Leu His Gly Gly Thr 4145 4150 4155 Cys Gln Gly Thr Arg Cys Leu Cys Leu Pro Gly Phe Ser Gly Pro 4160 4165 4170 Arg Cys Gln Gln Gly Ser Gly His Gly Ile Ala Glu Ser Asp Trp 4175 4180 4185 His Leu Glu Gly Ser Gly Gly Asn Asp Ala Pro Gly Gln Tyr Gly 4190 4195 4200 Ala Tyr Phe His Asp Asp Gly Phe Leu Ala Phe Pro Gly His Val 4205 4210 4215 Phe Ser Arg Ser Leu Pro Glu Val Pro Glu Thr Ile Glu Leu Glu 4220 4225 4230 Val Arg Thr Ser Thr Ala Ser Gly Leu Leu Leu Trp Gln Gly Val 4235 4240 4245 Glu Val Gly Glu Ala Gly Gln Gly Lys Asp Phe Ile Ser Leu Gly 4250 4255 4260 Leu Gln Asp Gly His Leu Val Phe Arg Tyr Gln Leu Gly Ser Gly 4265 4270 4275 Glu Ala Arg Leu Val Ser Glu Asp Pro Ile Asn Asp Gly Glu Trp 4280 4285 4290 His Arg Val Thr Ala Leu Arg Glu Gly Arg Arg Gly Ser Ile Gln 4295 4300 4305 Val Asp Gly Glu Glu Leu Val Ser Gly Arg Ser Pro Gly Pro Asn 4310 4315 4320 Val Ala Val Asn Ala Lys Gly Ser Val Tyr Ile Gly Gly Ala Pro 4325 4330 4335 Asp Val Ala Thr Leu Thr Gly Gly Arg Phe Ser Ser Gly Ile Thr 4340 4345 4350 Gly Cys Val Lys Asn Leu Val Leu His Ser Ala Arg Pro Gly Ala 4355 4360 4365 Pro Pro Pro Gln Pro Leu Asp Leu Gln His Arg Ala Gln Ala Gly 4370 4375 4380 Ala Asn Thr Arg Pro Cys Pro Ser 4385 4390 <210> SEQ ID NO 2 <211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Perlecan residues 2534-2551 Homo sapiens <400> SEQUENCE: 2 Tyr Pro Val Arg Ile Glu Ser Ser Ser Ala Ser Leu Ala Asn Gly His 1 5 10 15 Thr Leu <210> SEQ ID NO 3 <211> LENGTH: 347 <212> TYPE: PRT <213> ORGANISM: Decorin Homo sapiens <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: AAA52301 <309> DATABASE ENTRY DATE: 1994-11-07 <400> SEQUENCE: 3 Met Lys Ala Thr Ile Ile Leu Leu Leu Leu Ala Gln Val Ser Trp Ala 1 5 10 15 Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 20 25 30 Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro 35 40 45 Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 50 55 60 Val Gln Cys Ser Asp Leu Gly Leu Asp Lys Val Pro Lys Asp Leu Pro 65 70 75 80 Pro Asp Thr Thr Leu Leu Asp Leu Gln Asn Asn Lys Ile Thr Glu Ile 85 90 95 Lys Asp Gly Asp Phe Lys Asn Leu Lys Asn Leu His Ala Leu Ile Leu 100 105 110 Val Asn Asn Lys Ile Ser Lys Val Ser Pro Gly Ala Phe Thr Pro Leu 115 120 125 Val Lys Leu Glu Arg Leu Tyr Leu Ser Lys Asn Gln Leu Lys Glu Leu 130 135 140 Pro Glu Lys Met Pro Lys Thr Leu Gln Glu Leu Arg Ala His Glu Asn 145 150 155 160 Glu Ile Thr Lys Val Arg Lys Val Thr Phe Asn Gly Leu Asn Gln Met 165 170 175 Ile Val Ile Glu Leu Gly Thr Asn Pro Leu Lys Ser Ser Gly Ile Glu 180 185 190 Asn Gly Ala Phe Gln Gly Met Lys Lys Leu Ser Tyr Ile Arg Ile Ala 195 200 205 Asp Thr Asn Ile Thr Ser Ile Pro Gln Gly Leu Pro Pro Ser Leu Thr 210 215 220 Glu Leu His Leu Asp Gly Asn Lys Ile Ser Arg Val Asp Ala Ala Ser 225 230 235 240 Leu Lys Gly Leu Asn Asn Leu Ala Lys Leu Gly Leu Ser Phe Asn Ser 245 250 255 Ile Ser Ala Val Asp Asn Gly Ser Leu Ala Asn Thr Pro His Leu Arg 260 265 270 Glu Leu His Leu Asp Asn Asn Lys Leu Thr Arg Val Val Tyr Leu His 275 280 285 Asn Asn Asn Ile Ser Val Val Gly Ser Ser Asp Phe Cys Pro Pro Gly 290 295 300 His Asn Thr Lys Lys Ala Ser Tyr Ser Gly Val Ser Leu Phe Ser Asn 305 310 315 320 Pro Val Gln Tyr Trp Glu Ile Gln Pro Ser Thr Phe Arg Cys Val Tyr 325 330 335 Val Arg Ser Ala Ile Gln Leu Gly Asn Tyr Lys 340 345 <210> SEQ ID NO 4 <211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM: Decorin residues 207-230 Homo sapiens <400> SEQUENCE: 4 Ile Ala Asp Thr Asn Ile Thr Ser Ile Pro Gln Gly Leu Pro Pro Ser 1 5 10 15 Leu Thr Glu Leu Leu Asp Gly 20 <210> SEQ ID NO 5 <211> LENGTH: 368 <212> TYPE: PRT <213> ORGANISM: Biglycan Homo sapiens <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: AAH04244 <309> DATABASE ENTRY DATE: 2001-07-12 <400> SEQUENCE: 5 Met Trp Pro Leu Trp Arg Leu Val Ser Leu Leu Ala Leu Ser Gln Ala 1 5 10 15 Leu Pro Phe Glu Gln Arg Gly Phe Trp Asp Phe Thr Leu Asp Asp Gly 20 25 30 Pro Phe Met Met Asn Asp Glu Glu Ala Ser Gly Ala Asp Thr Ser Gly 35 40 45 Val Leu Asp Pro Asp Ser Val Thr Pro Thr Tyr Ser Ala Met Cys Pro 50 55 60 Phe Gly Cys His Cys His Leu Arg Val Val Gln Cys Ser Asp Leu Gly 65 70 75 80 Leu Lys Ser Val Pro Lys Glu Ile Ser Pro Asp Thr Thr Leu Leu Asp 85 90 95 Leu Gln Asn Asn Asp Ile Ser Glu Leu Arg Lys Asp Asp Phe Lys Gly 100 105 110 Leu Gln His Leu Tyr Ala Leu Val Leu Val Asn Asn Lys Ile Ser Lys 115 120 125 Ile His Glu Lys Ala Phe Ser Pro Leu Arg Lys Leu Gln Lys Leu Tyr 130 135 140 Ile Ser Lys Asn His Leu Val Glu Ile Pro Pro Asn Leu Pro Ser Ser 145 150 155 160 Leu Val Glu Leu Arg Ile His Asp Asn Arg Ile Arg Lys Val Pro Lys 165 170 175 Gly Val Phe Ser Gly Leu Arg Asn Met Asn Cys Ile Glu Met Gly Gly 180 185 190 Asn Pro Leu Glu Asn Ser Gly Phe Glu Pro Gly Ala Phe Asp Gly Leu 195 200 205 Lys Leu Asn Tyr Leu Arg Ile Ser Glu Ala Lys Leu Thr Gly Ile Pro 210 215 220 Lys Asp Leu Pro Glu Thr Leu Asn Glu Leu His Leu Asp His Asn Lys 225 230 235 240 Ile Gln Ala Ile Glu Leu Glu Asp Leu Leu Arg Tyr Ser Lys Leu Tyr 245 250 255 Arg Leu Gly Leu Gly His Asn Gln Ile Arg Met Ile Glu Asn Gly Ser 260 265 270 Leu Ser Phe Leu Pro Thr Leu Arg Glu Leu His Leu Asp Asn Asn Lys 275 280 285 Leu Ala Arg Val Pro Ser Gly Leu Pro Asp Leu Lys Leu Leu Gln Val 290 295 300 Val Tyr Leu His Ser Asn Asn Ile Thr Lys Val Gly Val Asn Asp Phe 305 310 315 320 Cys Pro Met Gly Phe Gly Val Lys Arg Ala Tyr Tyr Asn Gly Ile Ser 325 330 335 Leu Phe Asn Asn Pro Val Pro Tyr Trp Glu Val Gln Pro Ala Thr Phe 340 345 350 Arg Cys Val Thr Asp Arg Leu Ala Ile Gln Phe Gly Asn Tyr Lys Lys 355 360 365 <210> SEQ ID NO 6 <211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Biglycan residues 27-39 Homo sapiens <400> SEQUENCE: 6 Phe Thr Leu Asp Asp Gly Pro Phe Met Met Asn Asp Glu 1 5 10 <210> SEQ ID NO 7 <211> LENGTH: 935 <212> TYPE: PRT <213> ORGANISM: Protocadherin Homo sapiens <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: AAD43714 <309> DATABASE ENTRY DATE: 1999-07-22 <400> SEQUENCE: 7 Met Ala Asn Arg Leu Gln Arg Gly Asp Arg Ser Arg Leu Leu Leu Leu 1 5 10 15 Leu Cys Ile Phe Leu Gly Thr Leu Arg Gly Phe Arg Ala Arg Gln Ile 20 25 30 Arg Tyr Ser Val Pro Glu Glu Thr Glu Lys Gly Ser Phe Val Gly Asn 35 40 45 Ile Ser Lys Asp Leu Gly Leu Glu Pro Arg Glu Leu Ala Lys Arg Gly 50 55 60 Val Arg Ile Val Ser Arg Gly Lys Thr Gln Leu Phe Ala Val Asn Pro 65 70 75 80 Arg Ser Gly Ser Leu Ile Thr Ala Gly Arg Ile Asp Arg Glu Glu Leu 85 90 95 Cys Glu Thr Val Ser Ser Cys Phe Leu Asn Met Glu Leu Leu Val Glu 100 105 110 Asp Thr Leu Lys Ile Tyr Gly Val Glu Val Glu Ile Ile Asp Ile Asn 115 120 125 Asp Asn Ala Pro Ser Phe Gln Glu Asp Glu Val Glu Ile Lys Val Ser 130 135 140 Glu His Ala Ile Pro Gly Ala Arg Phe Ala Leu Pro Asn Ala Arg Asp 145 150 155 160 Pro Asp Val Gly Val Asn Ser Leu Gln Ser Tyr Gln Leu Ser Pro Asn 165 170 175 Asn Tyr Phe Ser Leu Gln Leu Arg Gly Arg Thr Asp Gly Ala Lys Asn 180 185 190 Pro Glu Leu Val Leu Glu Gly Ser Leu Asp Arg Glu Lys Glu Ala Ala 195 200 205 His Leu Leu Leu Leu Thr Ala Leu Asp Gly Gly Asp Pro Ile Arg Lys 210 215 220 Gly Ala Val Pro Ile Arg Val Val Val Leu Asp Val Asn Asp His Ile 225 230 235 240 Pro Met Phe Thr Gln Ser Val Tyr Arg Val Ser Val Pro Glu Asn Ile 245 250 255 Ser Ser Gly Thr Arg Val Leu Met Val Asn Ala Thr Asp Pro Asp Glu 260 265 270 Gly Ile Asn Gly Glu Val Met Tyr Ser Phe Arg Asn Met Glu Ser Lys 275 280 285 Ala Ser Glu Ile Phe Gln Leu Asp Ser Gln Thr Gly Glu Val Gln Val 290 295 300 Arg Gly Ser Leu Asp Phe Glu Lys Tyr Arg Phe Tyr Glu Met Glu Ile 305 310 315 320 Gln Gly Gln Asp Gly Gly Gly Leu Phe Thr Thr Thr Thr Met Leu Ile 325 330 335 Thr Val Val Asp Val Asn Asp Asn Ala Pro Glu Ile Thr Ile Thr Ser 340 345 350 Ser Ile Asn Ser Ile Leu Glu Asn Ser Pro Pro Gly Thr Val Ile Ala 355 360 365 Leu Leu Asn Val Gln Asp Gln Asp Ser Gly Glu Asn Gly Gln Val Ser 370 375 380 Cys Phe Ile Pro Asn His Leu Pro Phe Lys Leu Glu Lys Thr Tyr Gly 385 390 395 400 Asn Tyr Tyr Lys Leu Ile Thr Ser Arg Val Leu Asp Arg Glu Leu Val 405 410 415 Gln Ser Tyr Asn Ile Thr Leu Thr Ala Thr Asp Gln Gly Ser Pro Pro 420 425 430 Leu Ser Ala Glu Thr His Val Trp Leu Asn Val Ala Asp Asp Asn Asp 435 440 445 Asn Pro Pro Val Phe Pro His Ser Ser Tyr Ser Ala Tyr Ile Pro Glu 450 455 460 Asn Asn Pro Arg Gly Ala Ser Ile Phe Ser Val Thr Ala Leu Asp Pro 465 470 475 480 Asp Ser Lys Gln Asn Ala Leu Val Thr Tyr Ser Leu Thr Asp Asp Thr 485 490 495 Val Gln Gly Val Pro Leu Ser Ser Tyr Val Ser Ile Asn Ser Asn Thr 500 505 510 Gly Val Leu Tyr Ala Leu Gln Ser Phe Asp Tyr Glu Gln Phe Arg Asp 515 520 525 Leu Glu Leu Arg Val Ile Ala Arg Asp Ser Gly Asp Pro Pro Leu Ser 530 535 540 Ser Asn Val Ser Leu Ser Leu Phe Val Leu Asp Gln Asn Asp Asn Ala 545 550 555 560 Pro Glu Ile Leu Tyr Pro Ala Leu Pro Thr Asp Gly Ser Thr Gly Val 565 570 575 Glu Leu Ala Pro Arg Ser Ala Glu Pro Gly Tyr Leu Val Thr Lys Val 580 585 590 Val Ala Val Asp Lys Asp Ser Gly Gln Asn Ala Trp Leu Ser Tyr Arg 595 600 605 Leu Leu Lys Ala Ser Glu Pro Gly Leu Phe Ala Val Gly Glu His Thr 610 615 620 Gly Glu Val Arg Thr Ala Arg Ala Leu Leu Asp Arg Asp Ala Leu Lys 625 630 635 640 Gln Ser Leu Val Val Ala Val Gln Asp His Gly Gln Pro Pro Leu Ser 645 650 655 Ala Thr Val Thr Leu Thr Val Ala Val Ala Asp Ser Ile Pro Glu Val 660 665 670 Leu Ala Asp Leu Gly Ser Leu Glu Ser Leu Ala Asn Ser Glu Thr Ser 675 680 685 Asp Leu Ser Leu Tyr Leu Val Val Ala Val Ala Ala Val Ser Cys Ile 690 695 700 Phe Leu Val Phe Val Ile Val Leu Leu Ala Leu Arg Leu Trp Arg Trp 705 710 715 720 His Lys Ser Arg Leu Leu Gln Ala Ser Glu Gly Gly Leu Ala Gly Met 725 730 735 Pro Thr Ser His Phe Val Gly Val Asp Gly Val Gln Ala Phe Leu Gln 740 745 750 Thr Tyr Ser His Glu Val Ser Leu Ile Ala Asp Ser Gln Lys Ser His 755 760 765 Leu Ile Phe Pro Gln Pro Asn Tyr Gly Asp Thr Leu Ile Ser Gln Glu 770 775 780 Ser Cys Glu Lys Ser Glu Pro Leu Leu Ile Ala Glu Asp Ser Ala Ile 785 790 795 800 Ile Leu Gly Lys Cys Asp Pro Thr Ser Asn Gln Gln Ala Pro Pro Asn 805 810 815 Thr Asp Trp Arg Phe Ser Gln Ala Gln Arg Pro Gly Thr Ser Gly Ser 820 825 830 Gln Asn Gly Asp Asp Thr Gly Thr Trp Pro Asn Asn Gln Phe Asp Thr 835 840 845 Glu Met Leu Gln Ala Met Ile Leu Ala Ser Ala Ser Glu Ala Ala Asp 850 855 860 Gly Ser Ser Thr Leu Gly Gly Gly Ala Gly Thr Met Gly Leu Ser Ala 865 870 875 880 Arg Tyr Gly Pro Gln Phe Thr Leu Gln His Val Pro Asp Tyr Arg Gln 885 890 895 Asn Val Tyr Ile Pro Gly Ser Asn Ala Thr Leu Thr Asn Ala Ala Gly 900 905 910 Lys Arg Asp Gly Lys Ala Pro Ala Gly Gly Asn Gly Asn Lys Lys Lys 915 920 925 Ser Gly Lys Lys Glu Lys Lys 930 935 <210> SEQ ID NO 8 <211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: protocadherin residues 523-538 Homo sapiens <400> SEQUENCE: 8 Tyr Glu Gln Phe Arg Asp Leu Glu Leu Arg Val Ile Ala Arg Asp Ser 1 5 10 15 <210> SEQ ID NO 9 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: protocadherin residues 523-532 Homo sapiens <400> SEQUENCE: 9 Tyr Glu Gln Phe Arg Asp Leu Glu Leu Arg 1 5 10 <210> SEQ ID NO 10 <211> LENGTH: 3002 <212> TYPE: PRT <213> ORGANISM: Fibrillin-1 Homo sapiens <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER: A47221 <309> DATABASE ENTRY DATE: 2000-07-21 <400> SEQUENCE: 10 Tyr Phe Ser Arg Glu Lys Pro Leu Arg Gly Arg Tyr Leu Lys Arg Trp 1 5 10 15 Gly Lys Glu Gly Ala Ala Gly Ala Ala Ala Glu Thr Val Gly Ala Thr 20 25 30 Ser Gly Gln Glu Pro Gln Leu Gly Gln Leu Arg Ala Glu Pro Ser Ser 35 40 45 Gly Cys Ser Gly His Asp Trp Glu Gln Pro Pro Pro Pro Pro Arg Glu 50 55 60 Ser Glu Pro Pro Leu Leu His Trp Gln Gly Pro Pro Glu Val Gly Ala 65 70 75 80 Ala Pro Gly Glu Gly Gly Arg Ser Pro Ala Arg Gly Thr Gly Gly Gly 85 90 95 Ile Ala Gly Pro Arg Arg Arg Gly Ala Leu Gln Gly Ala Ala Ala Ala 100 105 110 Ala Asp Arg Ala Pro Gly Ala Ala Arg Gly Gly Gly Ser Arg Trp Arg 115 120 125 Leu Gly Ile Met Arg Arg Gly Arg Leu Leu Glu Ile Ala Leu Gly Phe 130 135 140 Thr Val Leu Leu Ala Ser Tyr Thr Ser His Gly Ala Asp Ala Asn Leu 145 150 155 160 Glu Ala Gly Asn Val Lys Glu Thr Arg Ala Ser Arg Ala Lys Arg Arg 165 170 175 Gly Gly Gly Gly His Asp Ala Leu Lys Gly Pro Asn Val Cys Gly Ser 180 185 190 Arg Tyr Asn Ala Tyr Cys Cys Pro Gly Trp Lys Thr Leu Pro Gly Gly 195 200 205 Asn Gln Cys Ile Val Pro Ile Cys Arg His Ser Cys Gly Asp Gly Phe 210 215 220 Cys Ser Arg Pro Asn Met Cys Thr Cys Pro Ser Gly Gln Ile Ala Pro 225 230 235 240 Ser Cys Gly Ser Arg Ser Ile Gln His Cys Asn Ile Arg Cys Met Asn 245 250 255 Gly Gly Ser Cys Ser Asp Asp His Cys Leu Cys Gln Lys Gly Tyr Ile 260 265 270 Gly Thr His Cys Gly Gln Pro Val Cys Glu Ser Gly Cys Leu Asn Gly 275 280 285 Gly Arg Cys Val Ala Pro Asn Arg Cys Ala Cys Thr Tyr Gly Phe Thr 290 295 300 Gly Pro Gln Cys Glu Arg Asp Tyr Arg Thr Gly Pro Cys Phe Thr Val 305 310 315 320 Ile Ser Asn Gln Met Cys Gln Gly Gln Leu Ser Gly Ile Val Cys Thr 325 330 335 Lys Gln Leu Cys Cys Ala Thr Val Gly Arg Ala Trp Gly His Pro Cys 340 345 350 Glu Met Cys Pro Ala Gln Pro His Pro Cys Arg Arg Gly Phe Ile Pro 355 360 365 Asn Ile Arg Thr Gly Ala Cys Gln Asp Val Asp Glu Cys Gln Ala Ile 370 375 380 Pro Gly Leu Cys Gln Gly Gly Asn Cys Ile Asn Thr Val Gly Ser Phe 385 390 395 400 Glu Cys Lys Cys Pro Ala Gly His Lys Leu Asn Glu Val Ser Gln Lys 405 410 415 Cys Glu Asp Ile Asp Glu Cys Ser Thr Ile Pro Gly Ile Cys Glu Gly 420 425 430 Gly Glu Cys Thr Asn Thr Val Ser Ser Tyr Phe Cys Lys Cys Pro Pro 435 440 445 Gly Phe Tyr Thr Ser Pro Asp Gly Thr Arg Cys Ile Asp Val Arg Pro 450 455 460 Gly Tyr Cys Tyr Thr Ala Leu Thr Asn Gly Arg Cys Ser Asn Gln Leu 465 470 475 480 Pro Gln Ser Ile Thr Lys Met Gln Cys Cys Cys Asp Ala Gly Arg Cys 485 490 495 Trp Ser Pro Gly Val Thr Val Ala Pro Glu Met Cys Pro Ile Arg Ala 500 505 510 Thr Glu Asp Phe Asn Lys Leu Cys Ser Val Pro Met Val Ile Pro Gly 515 520 525 Arg Pro Glu Tyr Pro Pro Pro Pro Leu Gly Pro Ile Pro Pro Val Leu 530 535 540 Pro Val Pro Pro Gly Phe Pro Pro Gly Pro Gln Ile Pro Val Pro Arg 545 550 555 560 Pro Pro Val Glu Tyr Leu Tyr Pro Ser Arg Glu Pro Pro Arg Val Leu 565 570 575 Pro Val Asn Val Thr Asp Tyr Cys Gln Leu Val Arg Tyr Leu Cys Gln 580 585 590 Asn Gly Arg Cys Ile Pro Thr Pro Gly Ser Tyr Arg Cys Glu Cys Asn 595 600 605 Lys Gly Phe Gln Leu Asp Leu Arg Gly Glu Cys Ile Asp Val Asp Glu 610 615 620 Cys Glu Lys Asn Pro Cys Ala Gly Gly Glu Cys Ile Asn Asn Gln Gly 625 630 635 640 Ser Tyr Thr Cys Gln Cys Arg Ala Gly Tyr Gln Ser Thr Leu Thr Arg 645 650 655 Thr Glu Cys Arg Asp Ile Asp Glu Cys Leu Gln Asn Gly Arg Ile Cys 660 665 670 Asn Asn Gly Arg Cys Ile Asn Thr Asp Gly Ser Phe His Cys Val Cys 675 680 685 Asn Ala Gly Phe His Val Thr Arg Asp Gly Lys Asn Cys Glu Asp Met 690 695 700 Asp Glu Cys Ser Ile Arg Asn Met Cys Leu Asn Gly Met Cys Ile Asn 705 710 715 720 Glu Asp Gly Ser Phe Lys Cys Ile Cys Lys Pro Gly Phe Gln Leu Ala 725 730 735 Ser Asp Gly Arg Tyr Cys Lys Asp Ile Asn Glu Cys Glu Thr Pro Gly 740 745 750 Ile Cys Met Asn Gly Arg Cys Val Asn Thr Asp Gly Ser Tyr Arg Cys 755 760 765 Glu Cys Phe Pro Gly Leu Ala Val Gly Leu Asp Gly Arg Val Cys Val 770 775 780 Asp Thr His Met Arg Ser Thr Cys Tyr Gly Gly Tyr Lys Arg Gly Gln 785 790 795 800 Cys Ile Lys Pro Leu Phe Gly Ala Val Thr Lys Ser Glu Cys Cys Cys 805 810 815 Ala Ser Thr Glu Tyr Ala Phe Gly Glu Pro Cys Gln Pro Cys Pro Ala 820 825 830 Gln Asn Ser Ala Glu Tyr Gln Ala Leu Cys Ser Ser Gly Pro Gly Met 835 840 845 Thr Ser Ala Gly Ser Asp Ile Asn Glu Cys Ala Leu Asp Pro Asp Ile 850 855 860 Cys Pro Asn Gly Ile Cys Glu Asn Leu Arg Gly Thr Tyr Lys Cys Ile 865 870 875 880 Cys Asn Ser Gly Tyr Glu Val Asp Ser Thr Gly Lys Asn Cys Val Asp 885 890 895 Ile Asn Glu Cys Val Leu Asn Ser Leu Leu Cys Asp Asn Gly Gln Cys 900 905 910 Arg Asn Thr Pro Gly Ser Phe Val Cys Thr Cys Pro Lys Gly Phe Ile 915 920 925 Tyr Lys Pro Asp Leu Lys Thr Cys Glu Asp Ile Asp Glu Cys Glu Ser 930 935 940 Ser Pro Cys Ile Asn Gly Val Cys Lys Asn Ser Pro Gly Ser Phe Ile 945 950 955 960 Cys Glu Cys Ser Ser Glu Ser Thr Leu Asp Pro Thr Lys Thr Ile Cys 965 970 975 Ile Glu Thr Ile Lys Gly Thr Cys Trp Gln Thr Val Ile Asp Gly Arg 980 985 990 Cys Glu Ile Asn Ile Asn Gly Ala Thr Leu Lys Ser Gln Cys Cys Ser 995 1000 1005 Ser Leu Gly Ala Ala Trp Gly Ser Pro Cys Thr Leu Cys Gln Val 1010 1015 1020 Asp Pro Ile Cys Gly Lys Gly Tyr Ser Arg Ile Lys Gly Thr Gln 1025 1030 1035 Cys Glu Asp Ile Asp Glu Cys Glu Val Phe Pro Gly Val Cys Lys 1040 1045 1050 Asn Gly Leu Cys Val Asn Thr Arg Gly Ser Phe Lys Cys Gln Cys 1055 1060 1065 Pro Ser Gly Met Thr Leu Asp Ala Thr Gly Arg Ile Cys Leu Asp 1070 1075 1080 Ile Arg Leu Glu Thr Cys Phe Leu Arg Tyr Glu Asp Glu Glu Cys 1085 1090 1095 Thr Leu Pro Ile Ala Gly Arg His Arg Met Asp Ala Cys Cys Cys 1100 1105 1110 Ser Val Gly Ala Ala Trp Gly Thr Glu Glu Cys Glu Glu Cys Pro 1115 1120 1125 Met Arg Asn Thr Pro Glu Tyr Glu Glu Leu Cys Pro Arg Gly Pro 1130 1135 1140 Gly Phe Ala Thr Lys Glu Ile Thr Asn Gly Lys Pro Phe Phe Lys 1145 1150 1155 Asp Ile Asn Glu Cys Lys Met Ile Pro Ser Leu Cys Thr His Gly 1160 1165 1170 Lys Cys Arg Asn Thr Ile Gly Ser Phe Lys Cys Arg Cys Asp Ser 1175 1180 1185 Gly Phe Ala Leu Asp Ser Glu Glu Arg Asn Cys Thr Asp Ile Asp 1190 1195 1200 Glu Cys Arg Ile Ser Pro Asp Leu Cys Gly Arg Gly Gln Cys Val 1205 1210 1215 Asn Thr Pro Gly Asp Phe Glu Cys Lys Cys Asp Glu Gly Tyr Glu 1220 1225 1230 Ser Gly Phe Met Met Met Lys Asn Cys Met Asp Ile Asp Glu Cys 1235 1240 1245 Gln Arg Asp Pro Leu Leu Cys Arg Gly Gly Val Cys His Asn Thr 1250 1255 1260 Glu Gly Ser Tyr Arg Cys Glu Cys Pro Pro Gly His Gln Leu Ser 1265 1270 1275 Pro Asn Ile Ser Ala Cys Ile Asp Ile Asn Glu Cys Glu Leu Ser 1280 1285 1290 Ala His Leu Cys Pro Asn Gly Arg Cys Val Asn Leu Ile Gly Lys 1295 1300 1305 Tyr Gln Cys Ala Cys Asn Pro Gly Tyr His Ser Thr Pro Asp Arg 1310 1315 1320 Leu Phe Cys Val Asp Ile Asp Glu Cys Ser Ile Met Asn Gly Gly 1325 1330 1335 Cys Glu Thr Phe Cys Thr Asn Ser Glu Gly Ser Tyr Glu Cys Ser 1340 1345 1350 Cys Gln Pro Gly Phe Ala Leu Met Pro Asp Gln Arg Ser Cys Thr 1355 1360 1365 Asp Ile Asp Glu Cys Glu Asp Asn Pro Asn Ile Cys Asp Gly Gly 1370 1375 1380 Gln Cys Thr Asn Ile Pro Gly Glu Tyr Arg Cys Leu Cys Tyr Asp 1385 1390 1395 Gly Phe Met Ala Ser Glu Asp Met Lys Thr Cys Val Asp Val Asn 1400 1405 1410 Glu Cys Asp Leu Asn Pro Asn Ile Cys Leu Ser Gly Thr Cys Glu 1415 1420 1425 Asn Thr Lys Gly Ser Phe Ile Cys His Cys Asp Met Gly Tyr Ser 1430 1435 1440 Gly Lys Lys Gly Lys Thr Gly Cys Thr Asp Ile Asn Glu Cys Glu 1445 1450 1455 Ile Gly Ala His Asn Cys Gly Lys His Ala Val Cys Thr Asn Thr 1460 1465 1470 Ala Gly Ser Phe Lys Cys Ser Cys Ser Pro Gly Trp Ile Gly Asp 1475 1480 1485 Gly Ile Lys Cys Thr Asp Leu Asp Glu Cys Ser Asn Gly Thr His 1490 1495 1500 Met Cys Ser Gln His Ala Asp Cys Lys Asn Thr Met Gly Ser Tyr 1505 1510 1515 Arg Cys Leu Cys Lys Glu Gly Tyr Thr Gly Asp Gly Phe Thr Cys 1520 1525 1530 Thr Asp Leu Asp Glu Cys Ser Glu Asn Leu Asn Leu Cys Gly Asn 1535 1540 1545 Gly Gln Cys Leu Asn Ala Pro Gly Gly Tyr Arg Cys Glu Cys Asp 1550 1555 1560 Met Gly Phe Val Pro Ser Ala Asp Gly Lys Ala Cys Glu Asp Ile 1565 1570 1575 Asp Glu Cys Ser Leu Pro Asn Ile Cys Val Phe Gly Thr Cys His 1580 1585 1590 Asn Leu Pro Gly Leu Phe Arg Cys Glu Cys Glu Ile Gly Tyr Glu 1595 1600 1605 Leu Asp Arg Ser Gly Gly Asn Cys Thr Asp Val Asn Glu Cys Leu 1610 1615 1620 Asp Pro Thr Thr Cys Ile Ser Gly Asn Cys Val Asn Thr Pro Gly 1625 1630 1635 Ser Tyr Ile Cys Asp Cys Pro Pro Asp Phe Glu Leu Asn Pro Thr 1640 1645 1650 Arg Val Gly Cys Val Asp Thr Arg Ser Gly Asn Cys Tyr Leu Asp 1655 1660 1665 Ile Arg Pro Arg Gly Asp Asn Gly Asp Thr Ala Cys Ser Asn Glu 1670 1675 1680 Ile Gly Val Gly Val Ser Lys Ala Ser Cys Cys Cys Ser Leu Gly 1685 1690 1695 Lys Ala Trp Gly Thr Pro Cys Glu Met Cys Pro Ala Val Asn Thr 1700 1705 1710 Ser Glu Tyr Lys Ile Leu Cys Pro Gly Gly Glu Gly Phe Arg Pro 1715 1720 1725 Asn Pro Ile Thr Val Ile Leu Glu Asp Ile Asp Glu Cys Gln Glu 1730 1735 1740 Leu Pro Gly Leu Cys Gln Gly Gly Lys Cys Ile Asn Thr Phe Gly 1745 1750 1755 Ser Phe Gln Cys Arg Cys Pro Thr Gly Tyr Tyr Leu Asn Glu Asp 1760 1765 1770 Thr Arg Val Cys Asp Asp Val Asn Glu Cys Glu Thr Pro Gly Ile 1775 1780 1785 Cys Gly Pro Gly Thr Cys Tyr Asn Thr Val Gly Asn Tyr Thr Cys 1790 1795 1800 Ile Cys Pro Pro Asp Tyr Met Gln Val Asn Gly Gly Asn Asn Cys 1805 1810 1815 Met Asp Met Arg Arg Ser Leu Cys Tyr Arg Asn Tyr Tyr Ala Asp 1820 1825 1830 Asn Gln Thr Cys Asp Gly Glu Leu Leu Phe Asn Met Thr Lys Lys 1835 1840 1845 Met Cys Cys Cys Ser Tyr Asn Ile Gly Arg Ala Trp Asn Lys Pro 1850 1855 1860 Cys Glu Gln Cys Pro Ile Pro Ser Thr Asp Glu Phe Ala Thr Leu 1865 1870 1875 Cys Gly Ser Gln Arg Pro Gly Phe Val Ile Asp Ile Tyr Thr Gly 1880 1885 1890 Leu Pro Val Asp Ile Asp Glu Cys Arg Glu Ile Pro Gly Val Cys 1895 1900 1905 Glu Asn Gly Val Cys Ile Asn Met Val Gly Ser Phe Arg Cys Glu 1910 1915 1920 Cys Pro Val Gly Phe Phe Tyr Asn Asp Lys Leu Leu Val Cys Glu 1925 1930 1935 Asp Ile Asp Glu Cys Gln Asn Gly Pro Val Cys Gln Arg Asn Ala 1940 1945 1950 Glu Cys Ile Asn Thr Ala Gly Ser Tyr Arg Cys Asp Cys Lys Pro 1955 1960 1965 Gly Tyr Arg Phe Thr Ser Thr Gly Gln Cys Asn Asp Arg Asn Glu 1970 1975 1980 Cys Gln Glu Ile Pro Asn Ile Cys Ser His Gly Gln Cys Ile Asp 1985 1990 1995 Thr Val Gly Ser Phe Tyr Cys Leu Cys His Thr Gly Phe Lys Thr 2000 2005 2010 Asn Asp Asp Gln Thr Met Cys Leu Asp Ile Asn Glu Cys Glu Arg 2015 2020 2025 Asp Ala Cys Gly Asn Gly Thr Cys Arg Asn Thr Ile Gly Ser Phe 2030 2035 2040 Asn Cys Arg Cys Asn His Gly Phe Ile Leu Ser His Asn Asn Asp 2045 2050 2055 Cys Ile Asp Val Asp Glu Cys Ala Ser Gly Asn Gly Asn Leu Cys 2060 2065 2070 Arg Asn Gly Gln Cys Ile Asn Thr Val Gly Ser Phe Gln Cys Gln 2075 2080 2085 Cys Asn Glu Gly Tyr Glu Val Ala Pro Asp Gly Arg Thr Cys Val 2090 2095 2100 Asp Ile Asn Glu Cys Leu Leu Glu Pro Arg Lys Cys Ala Pro Gly 2105 2110 2115 Thr Cys Gln Asn Leu Asp Gly Ser Tyr Arg Cys Ile Cys Pro Pro 2120 2125 2130 Gly Tyr Ser Leu Gln Asn Glu Lys Cys Glu Asp Ile Asp Glu Cys 2135 2140 2145 Val Glu Glu Pro Glu Ile Cys Ala Leu Gly Thr Cys Ser Asn Thr 2150 2155 2160 Glu Gly Ser Phe Lys Cys Leu Cys Pro Glu Gly Phe Ser Leu Ser 2165 2170 2175 Ser Ser Gly Arg Arg Cys Gln Asp Leu Arg Met Ser Tyr Cys Tyr 2180 2185 2190 Ala Lys Phe Glu Gly Gly Lys Cys Ser Ser Pro Lys Ser Arg Asn 2195 2200 2205 His Ser Lys Gln Glu Cys Cys Cys Ala Leu Lys Gly Glu Gly Trp 2210 2215 2220 Gly Asp Pro Cys Glu Leu Cys Pro Thr Glu Pro Asp Glu Ala Phe 2225 2230 2235 Arg Gln Ile Cys Pro Tyr Gly Ser Gly Ile Ile Val Gly Pro Asp 2240 2245 2250 Asp Ser Ala Val Asp Met Asp Glu Cys Lys Glu Pro Asp Val Cys 2255 2260 2265 Lys His Gly Gln Cys Ile Asn Thr Asp Gly Ser Tyr Arg Cys Glu 2270 2275 2280 Cys Pro Phe Gly Tyr Thr Leu Ala Gly Asn Glu Cys Val Asp Thr 2285 2290 2295 Asp Glu Cys Ser Val Gly Asn Pro Cys Gly Asn Gly Thr Cys Lys 2300 2305 2310 Asn Val Ile Gly Gly Phe Glu Cys Thr Cys Glu Glu Gly Phe Glu 2315 2320 2325 Pro Gly Pro Met Met Thr Cys Glu Asp Ile Asn Glu Cys Ala Gln 2330 2335 2340 Asn Pro Leu Leu Cys Ala Phe Arg Cys Val Asn Thr Tyr Gly Ser 2345 2350 2355 Tyr Glu Cys Lys Cys Pro Val Gly Tyr Val Leu Arg Glu Asp Arg 2360 2365 2370 Arg Met Cys Lys Asp Glu Asp Glu Cys Glu Glu Gly Lys His Asp 2375 2380 2385 Cys Thr Glu Lys Gln Met Glu Cys Lys Asn Leu Ile Gly Thr Tyr 2390 2395 2400 Met Cys Ile Cys Gly Pro Gly Tyr Gln Arg Arg Pro Asp Gly Glu 2405 2410 2415 Gly Cys Val Asp Glu Asn Glu Cys Gln Thr Lys Pro Gly Ile Cys 2420 2425 2430 Glu Asn Gly Arg Cys Leu Asn Thr Arg Gly Ser Tyr Thr Cys Glu 2435 2440 2445 Cys Asn Asp Gly Phe Thr Ala Ser Pro Asn Gln Asp Glu Cys Leu 2450 2455 2460 Asp Asn Arg Glu Gly Tyr Cys Phe Thr Glu Val Leu Gln Asn Met 2465 2470 2475 Cys Gln Ile Gly Ser Ser Asn Arg Asn Pro Val Thr Lys Ser Glu 2480 2485 2490 Cys Cys Cys Asp Gly Gly Arg Gly Trp Gly Pro His Cys Glu Ile 2495 2500 2505 Cys Pro Phe Gln Gly Thr Val Ala Phe Lys Lys Leu Cys Pro His 2510 2515 2520 Gly Arg Gly Phe Met Thr Asn Gly Ala Asp Ile Asp Glu Cys Lys 2525 2530 2535 Val Ile His Asp Val Cys Arg Asn Gly Glu Cys Val Asn Asp Arg 2540 2545 2550 Gly Ser Tyr His Cys Ile Cys Lys Thr Gly Tyr Thr Pro Asp Ile 2555 2560 2565 Thr Gly Thr Ser Cys Val Asp Leu Asn Glu Cys Asn Gln Ala Pro 2570 2575 2580 Lys Pro Cys Asn Phe Ile Cys Lys Asn Thr Glu Gly Ser Tyr Gln 2585 2590 2595 Cys Ser Cys Pro Lys Gly Tyr Ile Leu Gln Glu Asp Gly Arg Ser 2600 2605 2610 Cys Lys Asp Leu Asp Glu Cys Ala Thr Lys Gln His Asn Cys Gln 2615 2620 2625 Phe Leu Cys Val Asn Thr Ile Gly Gly Phe Thr Cys Lys Cys Pro 2630 2635 2640 Pro Gly Phe Thr Gln His His Thr Ser Cys Ile Asp Asn Asn Glu 2645 2650 2655 Cys Thr Ser Asp Ile Asn Leu Cys Gly Ser Lys Gly Ile Cys Gln 2660 2665 2670 Asn Thr Pro Gly Ser Phe Thr Cys Glu Cys Gln Arg Gly Phe Ser 2675 2680 2685 Leu Asp Gln Thr Gly Ser Ser Cys Glu Asp Val Asp Glu Cys Glu 2690 2695 2700 Gly Asn His Arg Cys Gln His Gly Cys Gln Asn Ile Ile Gly Gly 2705 2710 2715 Tyr Arg Cys Ser Cys Pro Gln Gly Tyr Leu Gln His Tyr Gln Trp 2720 2725 2730 Asn Gln Cys Val Asp Glu Asn Glu Cys Leu Ser Ala His Ile Cys 2735 2740 2745 Gly Gly Ala Ser Cys His Asn Thr Leu Gly Ser Tyr Lys Cys Met 2750 2755 2760 Cys Pro Ala Gly Phe Gln Tyr Glu Gln Phe Ser Gly Gly Cys Gln 2765 2770 2775 Asp Ile Asn Glu Cys Gly Ser Ala Gln Ala Pro Cys Ser Tyr Gly 2780 2785 2790 Cys Ser Asn Thr Glu Gly Gly Tyr Leu Cys Gly Cys Pro Pro Gly 2795 2800 2805 Tyr Phe Arg Ile Gly Gln Gly His Cys Val Ser Gly Met Gly Met 2810 2815 2820 Gly Arg Gly Asn Pro Glu Pro Pro Val Ser Gly Glu Met Asp Asp 2825 2830 2835 Asn Ser Leu Ser Pro Glu Ala Cys Tyr Glu Cys Lys Ile Asn Gly 2840 2845 2850 Tyr Pro Lys Arg Gly Arg Lys Arg Arg Ser Thr Asn Glu Thr Asp 2855 2860 2865 Ala Ser Asn Ile Glu Asp Gln Ser Glu Thr Glu Ala Asn Val Ser 2870 2875 2880 Leu Ala Ser Trp Asp Val Glu Lys Thr Ala Ile Phe Ala Phe Asn 2885 2890 2895 Ile Ser His Val Ser Asn Lys Val Arg Ile Leu Glu Leu Leu Pro 2900 2905 2910 Ala Leu Thr Thr Leu Thr Asn His Asn Arg Tyr Leu Ile Glu Ser 2915 2920 2925 Gly Asn Glu Asp Gly Phe Phe Lys Ile Asn Gln Lys Glu Gly Ile 2930 2935 2940 Ser Tyr Leu His Phe Thr Lys Lys Lys Pro Val Ala Gly Thr Tyr 2945 2950 2955 Ser Leu Gln Ile Ser Ser Thr Pro Leu Tyr Lys Lys Lys Glu Leu 2960 2965 2970 Asn Gln Leu Glu Asp Lys Tyr Asp Lys Asp Tyr Leu Ser Gly Glu 2975 2980 2985 Leu Gly Asp Asn Leu Lys Met Lys Ile Gln Val Leu Leu His 2990 2995 3000 <210> SEQ ID NO 11 <211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Fibrillin-1 residues 2270-2782 Homo sapiens <400> SEQUENCE: 11 Tyr Glu Gln Phe Ser Gly Gly Cys Gln Asp Ile Asn Glu 1 5 10 <210> SEQ ID NO 12 <211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 12 Leu Tyr Pro Asn Gln Thr Gly Leu Pro Asp Pro Leu Ser Arg 1 5 10 <210> SEQ ID NO 13 <211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 13 Ser Ala Ile Ile Ala Thr Glu Gln Leu Gln Ala Ala Tyr Glu Asp Gly 1 5 10 15 Phe His Gln Cys 20 <210> SEQ ID NO 14 <211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 14 Leu Ala Thr Thr Gly Gln Leu Tyr Leu Ala Trp Gln Ala Gly Met Asp 1 5 10 15 Met <210> SEQ ID NO 15 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 15 Thr Gly Glu Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val 1 5 10 15 <210> SEQ ID NO 16 <211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 16 Thr Gly Glu Asp Phe Val Asp Ile Pro Glu Asn 1 5 10 <210> SEQ ID NO 17 <211> LENGTH: 29 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 17 Val Ser Leu Pro Asn Tyr Pro Ala Ile Pro Ser Asp Ala Thr Leu Glu 1 5 10 15 Val Gln Ser Leu Arg Ser Asn Asp Ser Gly Val Tyr Arg 20 25 <210> SEQ ID NO 18 <211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION: (4)..(10) <223> OTHER INFORMATION: Xaa = any amino acid <400> SEQUENCE: 18 Glu Gly Ser Xaa Gly Ala Asp Gly Pro Xaa Gly Arg Asp Gly 1 5 10 <210> SEQ ID NO 19 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 19 Ala Gln Glu Asp Ser Asp His 1 5 <210> SEQ ID NO 20 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 20 Leu Leu Thr Gln Thr Asp Ser Asp Gly Arg 1 5 10 

1. A method of assay, comprising measuring in a biological sample the amount or presence of an isomerised or optically inverted protein or of one or more isomerised or optically inverted fragments from such a protein, wherein said protein is perlecan, biglycan, decorin, fibrillin-1, or protocadherin.
 2. A method as claimed in claim 1, which determines the amount or presence of at least one *Asx or *Glx containing protein or protein fragment in said biological sample, wherein *Asx is αD Asp, αD Asn, βL Asp or βD Asp and *Glx is αD Glu, αD Gln, γL Glu or γD Glu.
 3. A method as claimed in claim 2, wherein is measured the amount of at least one protein or protein fragment containing the perlecan derived amino acid sequence YPVRIESSSASLANGHTL (SEQ ID NO 2) or a fragment thereof containing the E and/or N residue, wherein N denotes αL-Asn or an amino acid covered by the term *Asx and E denotes αL-Glu or an amino acid covered by the term *Glx.
 4. A method as claimed in claim 2, wherein is measured the amount of at least one protein or protein fragment containing the decorin derived amino acid sequence IADTNITSIPQGLPPSLTELLDG (SEQ ID NO 4) or a fragment thereof containing the D, E, N and/or Q residue, wherein N denotes αL-Asn or an amino acid covered by the term *Asx, D denotes αL-Asp or an amino acid covered by the term *Asx, E denotes αL-Glu or an amino acid covered by the term *Glx and Q denotes αL-Gln or an amino acid covered by the term *Glx.
 5. A method as claimed in claim 2, wherein is measured the amount of at least one protein or protein fragment containing the biglycan derived amino acid sequence FTLDDGPFMMNDE (SEQ ID NO 6) or a fragment thereof containing the D and/or E residue, wherein D denotes αL-Asp or an amino acid covered by the term *Asx and E denotes αL-Glu or an amino acid covered by the term *Glx.
 6. A method as claimed in claim 2, wherein is measured the amount of at least one protein or protein fragment containing the protocadherin derived amino acid sequence YEQFRDLELRVIARDS (SEQ ID NO 8) or a fragment thereof containing the D and/or E residue, wherein D denotes αL-Asp or an amino acid covered by the term *Asx and E denotes αL-Glu or an amino acid covered by the term *Glx.
 7. A method as claimed in claim 2, wherein is measured the amount of at least one protein or protein fragment containing the fibrillin-1 derived amino acid sequence YEQFSGGCBQDINE (SEQ ID NO 11) or a fragment thereof containing the D, E, N and/or Q residue, wherein N denotes αL-Asn or an amino acid covered by the term *Asx, D denotes αL-Asp or an amino acid covered by the term *Asx, E denotes αL-Glu or an amino acid covered by the term *Glx and Q denotes αL-Gln or an amino acid covered by the term *Glx.
 8. A method as claimed in claim 2, wherein is measured the amount of at least one protein or protein fragment containing the protocadherin derived amino acid sequence YEQFRDLELR (SEQ ID NO 9) or a fragment thereof containing the D and/or E residue, wherein D denotes αL-Asp or an amino acid covered by the term *Asx and E denotes αL-Glu or an amino acid covered by the term *Glx.
 9. A method of assay, comprising measuring in a sample the amount or presence of an isomerised or optically inverted fragments of a protein, wherein (a) said protein is aggrecan and said fragment contains one of the amino acid sequences: LYPNQTGLPDPLSR (SEQ ID NO 12), SAIIATEQLQAAYEDGFHQC (SEQ ID NO 13), LATTGQLYLAWQAGMDM (SEQ ID NO 14), TGEDFVDIPENFFGV (SEQ ID NO 15), TGEDFVDIPEN (SEQ ID NO 16) or VSLPNYPAIPSDATLEVQSLRSNDSGVYR (SEQ ID NO 17) or a fragment thereof containing the D, E, N and/or Q residue, wherein N denotes αL-Asn or an amino acid covered by the term *Asx, D denotes αL-Asp or an amino acid covered by the term *Asx, E denotes αL-Glu or an amino acid covered by the term *Glx and Q denotes αL-Gln or an amino acid covered by the term *Glx; (b) said protein is type II collagen and said fragment contains the amino acid sequence EGSXGADGPXGRDG (SEQ ID NO 18) or a fragment thereof containing the D and/or E residue, wherein D denotes αL-Asp or an amino acid covered by the term *Asx and E denotes αL-Glu or an amino acid covered by the term *Glx; (c) said protein is COMP and said fragment contains the amino acid sequence AQEDSDH (SEQ ID NO 19) or a fragment thereof containing the D, E and/or Q residue, wherein D denotes αL-Asp or an amino acid covered by the term *Asx, E denotes αL-Glu or an amino acid covered by the term *Glx and Q denotes αL-Gln or an amino acid covered by the term *Glx; or (d) said protein is CILP and said fragment contains the amino acid sequence LLTQTDSDGR (SEQ ID NO 20) or a fragment thereof containing the D and/or Q residue, wherein D denotes αL-Asp or an amino acid covered by the term *Asx and Q denotes αL-Gln or an amino acid covered by the term *Glx.
 10. A method as claimed in claim 1, wherein said measurement is carried out using an immunological binding partner which specifically binds an amino acid sequence comprising *Asx or *Glx.
 11. A method as claimed in claim 10, wherein said immunological binding partner is an antibody raised against a synthetic peptide having an amino acid sequence comprising *Asx or *Glx, or fragment of such an antibody having immunological binding specificity.
 12. A method as claimed in claim 10, wherein said amino acid sequence corresponds to a characteristic sequence of said protein, with *Asx or *Glx substituting for αL-Asp, -Asn, -Gln, or -Glu in said protein sequence.
 13. A method as claimed in claim 1, wherein said measurement provides an index of cartilage turnover relevant for conditions and diseases affecting joint tissue turnover.
 14. A method as claimed in claim 13, further comprising carrying out a measurement of a second index of joint disease and determining the value of a parameter mathematically combining said two indices.
 15. The use of an isomerised or optically inverted protein or of one or more isomerised or optically inverted fragments from such a protein in an in vitro method of assay for use in the diagnosis or the assessment of the severity of OA or RA, wherein said protein is perlecan, biglycan, decorin, fibrillin-1, or protocadherin.
 16. The use of an immunological binding partner which specifically binds an amino acid sequence comprising *Asx or *Glx flanked by amino acid residues of perlecan, biglycan, decorin, fibrillin-1, or protocadherin in an in vitro method for use in the diagnosis or the assessment of the severity of OA or RA.
 17. An immunological binding partner which specifically binds an amino acid sequence comprising *Asx or *Glx flanked by amino acid residues of perlecan, biglycan, decorin, fibrillin-1, or protocadherin.
 18. An immunological binding partner, which specifically binds the perlecan derived amino acid sequence YPVRIESSSASLANGHTL (SEQ ID NO 2) or a fragment thereof containing the E and/or N residue, wherein N denotes αL-Asn or an amino acid covered by the term *Asx and E denotes αL-Glu or an amino acid covered by the term *Glx, or the decorin derived amino acid sequence LADTNITSIPOGLPPSLTELLDG (SEQ ID NO 4) or a fragment thereof containing the D, E, N and/or Q residue, wherein N denotes αL-Asn or an amino acid covered by the term *Asx, D denotes αL-Asp or an amino acid covered by the term *Asx, E denotes αL-Glu or an amino acid covered by the term *Glx and Q denotes αL-Gln or an amino acid covered by the term *Glx, or the biglycan derived amino acid sequence FTLDDGPFMMNDE (SEQ ID NO 6) or a fragment thereof containing the D and/or E residue, wherein D denotes αL-Asp or an amino acid covered by the term *Asx and E denotes αL-Glu or an amino acid covered by the term *Glx, or the protocadherin derived amino acid sequence YEOFRDLELRVIARDS (SEQ ID NO 8) or a fragment thereof containing the D and/or E residue, wherein D denotes αL-Asp or an amino acid covered by the term *Asx and E denotes αL-Glu or an amino acid covered by the term *Glx, or the fibrillin-1 derived amino acid sequence YEQFSGGCBODINE (SEQ ID NO 11) or a fragment thereof containing the D, E, N and/or Q residue, wherein N denotes αL-Asn or an amino acid covered by the term *Asx, D denotes αL-Asp or an amino acid covered by the term *Asx, E denotes αL-Glu or an amino acid covered by the term *Glx and Q denotes αL-Gln or an amino acid covered by the term *Glx, or the protocadherin derived amino acid sequence YEQFRDLELR (SEQ ID NO 9) or a fragment thereof containing the D and/or E residue, wherein D denotes αL-Asp or an amino acid covered by the term *Asx and E denotes αL-Glu or an amino acid covered by the term *Glx, or one of the amino acid sequences: LYPNOTGLPDPLSR (SEQ ID NO 12) SAIIATEOLQAAYEDGFHOC (SEQ ID NO 13), LATTGOLYLAWOAGMDM (SEQ ID NO 14), TGEDFVDIPENFFGV (SEQ ID NO 15), TGEDFVDIPEN (SEQ ID NO 16) or VSLPNYPAIPSDATLEVOSLRSNDSGVYR (SEQ ID NO 17) or a fragment thereof containing the D, E, N and/or Q residue, wherein N denotes αL-Asn or an amino acid covered by the term *Asx, D denotes αL-Asp or an amino acid covered by the term *Asx, E denotes αL-Glu or an amino acid covered by the term *Glx and Q denotes αL-Gln or an amino acid covered by the term *Glx, or the amino acid sequence EGSXGADGPXGRDG (SEQ ID NO 18) or a fragment thereof containing the D and/or E residue, wherein D denotes αL-Asp or an amino acid covered by the term *Asx and E denotes αL-Glu or an amino acid covered by the term *Glx, or the amino acid sequence AOEDSDH (SEQ ID NO 19) or a fragment thereof containing the D, E and/or Q residue, wherein D denotes αL-Asp or an amino acid covered by the term *Asx, E denotes αL-Glu or an amino acid covered by the term *Glx and Q denotes αL-Gln or an amino acid covered by the term *Glx; or the amino acid sequence LLTOTDSDGR (SEQ ID NO 20) or a fragment thereof containing the D and/or Q residue, wherein D denotes αL-Asp or an amino acid covered by the term *Asx and Q denotes αL-Gln or an amino acid covered by the term *Glx.
 19. A cell line producing a monoclonal antibody, which is an immunological binding partner as claimed in claim
 17. 20. A peptide of up to 20 amino acids in length containing *Asx or *Glx flanked by amino acid residues of pertelcan, biglycan, decorin, fibrillin-1, or proto-cadherin.
 21. A peptide of up to 20 amino acids in length containing an N or a D of one of the sequences set out in claim 9 within a subsequence within said peptide comprising at least four contiguous amino acids of a said sequence set out in claim
 9. 22. The use in an assay for protein or protein fragments of a peptide as claimed in claim
 20. 23. A method of immunoassay in which a biological sample is contacted with an immunological binding agent in the presence of a peptide as claimed in claim 20 acting as a competition agent for binding to said immunological binding agent.
 24. A test kit comprising (a) an immunological binding partner as claimed in claim 17, or (b) a peptide as claimed in claim 20, in combination the other of (a) or (b), and optionally in combination with one or more of apparatus in which to perform an immunoassay, an antibody-enzyme conjugate, a substrate for an enzyme component of an antibody-enzyme conjugate, an enzyme-substrate reaction stopping composition, or a wash solution, a carrier bound to said binding partner or a detectable label bound to said binding partner.
 25. A method of therapeutic intervention for treating a pathological condition in a mammal comprising administrating a peptide as specified in claim
 20. 